Dipole electrical stimulation employing direct current for recovery from spinal cord injury

ABSTRACT

A system and method to treat neuromuscular conditions, including spinal cord injury, by what is characterized as dipole (two point) cortico-muscular stimulation. Two-point stimulation, with oppositely charged electrodes, allows pulsed, direct current to pass through the cortico-muscular pathway. The electrodes are placed on nerves, muscles, or a combination of both, that are on opposite sides of the spinal column, forming a current that passes across the spinal column. Further, an active electrode can be placed on the spinal column and a reference electrode can be placed outside the central nervous system. These methods improve functional recovery of the motor pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 61/253,948, filed on Oct. 22, 2009 and U.S.Provisional Application Ser. No. 61/316,319, filed on Mar. 22, 2010, theentire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with support under New York State Department ofHealth (NYS/DOH) grant No. CO23684 and Professional Staff Congress atthe City University of New York (PSCCUNY) grant No. 60027-37-39. Thestate of New York has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to the field of providingstimulation of central nervous system tissue, muscles, nerves, orcombinations thereof, and more particularly to a system and method fortreating neuromuscular conditions through two-point electricalstimulation.

BACKGROUND OF THE INVENTION

The nervous system comprises the central and the peripheral nervoussystem. The central nervous system is composed of the brain and thespinal cord, and the peripheral nervous system consists of all of theother neural elements, namely the nerves and ganglia outside of thebrain and spinal cord.

Damage to the nervous system may result from a traumatic injury, such aspenetrating trauma or blunt trauma, or a disease or disorder including,but not limited to Alzheimers disease, multiple sclerosis, Huntington'sdisease. amyotrophic lateral sclerosis (ALS), diabetic neuropathy,senile dementia, stroke and ischemia.

After spinal cord injury (SCI), spared regions of the central nervoussystem are spontaneously capable of repairing the damaged pathway,although the process is very limited. Moreover, despite the manypromising treatment strategies to improve connections across the damagedspinal cord, the strength of connectivity and functional recovery of theimpaired spinal cord are still unsatisfactory. It is well known thatspared axons sprout after SCI. See Murray M., Goldberger M. E.,Restitution of function and collateral sprouting in the cat spinal cord:the partially hemisected animal, J. Comp. Neurol., 158(1):19-36 (1974);Bareyre F. M., Kerschensteiner M., Raineteau O., Mettenleiter T. C.,Weinmann O., Schwab M. E., The injured spinal cord spontaneously forms anew intraspinal circuit in adult rats, Nat. Neurosci. 7:269-77 (2004);Brus-Ramer M., Carmel J. B., Chakrabarty S., Martin J. H., Electricalstimulation of spared corticospinal axons augments connections withipsilateral spinal motor circuits after injury, J. Neurosci.27:13793-13901 (2007). But fine-tuning of the process of sprouting ofspared axons after SCI as well as synapse stabilization might bedependent on precise pathway-selective activity.

Electrical stimulation of the central and peripheral nervous systemsimproves neuronal connectivity, and can be employed used to improvefunctional recovery after neuronal injury. It is an effective methodthat promotes reactive sprouting through which an increase in the numberof functional connections may be possible. Electrical stimulation canalso improve functional connections by strengthening the weak existingsynapses and/or by promoting synaptogenesis. One of the emergingconcepts is that the nervous system contains latent pathways that can beawoken by electrical stimulation or pharmacological manipulation.

The majority of the methods employing electrical stimulation utilize aone-point experimental paradigm in which unipolar or bipolar stimuli aredelivered at one point of the sensorimotor pathway. The effectiveness ofthis stimulation depends on active propagation of an action potentialthrough spared axons. Practically, one-point stimulation would be onlyeffective if the neuronal connections exist and can support active andsuccessful propagation of generated potentials. Therefore, one-pointstimulation would be restricted in its efficacy and inclined towardstronger connections.

The loss of neuromuscular activity after SCI leads to inevitableabnormalities that limit the effectiveness of one-point stimulation byblocking excitatory responses from traveling across the sensorimotorpathway. Some of these abnormalities are muscle atrophy and peripheralnerve inexcitability. In addition, changes of the sensorimotor pathwaybelow and above the lesion may involve several different mechanisms;some of them may be maladaptative. This maladaptive function will biasstimuli toward connections with better integrity, further limiting theeffectiveness of localized stimulation.

According to the Habbian plasticity principle, physiological processesstrengthen synaptic connections when presynaptic activity correlateswith postsynaptic firing. See, for example, Hebb D, Organization ofBehavior, New York, Wiley (1949). This phenomenon is known as long termpotentiation (“LTP”). LTP could be induced by high-frequency presynapticstimulation or by pairing low-frequency stimulation with postsynapticdepolarization. LTP can also be induced if a pre-synaptic input isactivated concurrently with post-synaptic input. In addition, directcurrent passed through a neuronal pathway can modulate the excitabilityof that pathway depending on the current polarity and neuronal geometry.In that, anodal stimulation would excite while cathodal stimulationinhibits neuronal activity.

Thus, there is a great desire to improve the effectiveness of electricalstimulation when treating neuromuscular conditions, such as SCI.

SUMMARY OF THE INVENTION

A system and method for treating neuromuscular conditions includingspinal cord injury (SCI) are disclosed herein, which employ dipole (twopoint) cortico-muscular stimulation (“dCMS”). The system and methodadvantageously employ a discovery that utilizing a two point method ofcortico-muscular stimulation dramatically improves the performance ofthe skeletal muscles after SCI. Two-point stimulation, with oppositelycharged electrodes, allows pulsed, direct current to pass through thecortico-muscular pathway regardless of the extent of injury and numberof spared neurons. These methods enable permanent functional recovery ofthe motor pathway.

The research leading to the present invention has demonstrated that dCMSsubstantially improved muscle twitch force and spinal cord responses incontrol and SCI animals, and that passage of pulsed direct currentacross the cortico-muscular pathway promotes stronger connectionsbetween spinal motor circuits and the motor cortex.

In one exemplary embodiment, the dCMS configuration involves an activeelectrode at a first point and a reference electrode at a second point,and is situated such that the current passes across the spinal cord. Thefirst and second points may be the motor cortex, muscles, or acombination thereof. In one configuration, the active electrode can besituated at the motor cortex and the reference electrode is at acontralateral muscle (e.g., a partially isolated gastrocnemius muscle).The passage of pulsed direct current across the cortico-muscular pathwayovercomes many of the excitability-confounding factors previouslydescribed. The advantage of using this method of stimulation is that thepassage of such current will not be dependent on factors affected bylesion such as the degree of myelination, density of existingconnections, synaptic strength and/or general excitability of thesensorimotor pathway. Instead, the current will passively flow along thecore conductors of the target neuronal pathway leading to the activationof the entire pathway regardless of its morphological integrity. Dipolarcortico-muscular stimulation substantially improves muscle twitch forceand spinal cord responses.

In another exemplary embodiment, electrical pulses can be applied to thespinal column employing an active electrode applied thereto. At leastone reference electrode is applied to a point outside the centralnervous system. The electrical pulses can be employed alone, or incombination with additional electrical pulses applied to the motorcortex and/or at least one muscle to enhance neuromuscular performance.Multiple stimulator units can be used synchronously or asynchronously totreat various traumas and/or injuries to the nervous system that arepresent in the brain, spinal column, or in the peripheral nerves.

The use of dCMS enhances the excitability of the cortico-muscularconnections and can be used in human patients suffering from not justspinal cord injury, but also stroke, multiple sclerosis, and the like.Even humans who engage in athletic activity may use dCMS to enhanceneuromuscular performance. It can practically be employed to strengthenor awaken any weak or dormant pathway in the nervous system.

According to another aspect of the present invention, a method ofimproving a neuromuscular condition of a vertebrate being is provided.The method includes: placing at least one active electrode at, or inproximity to, or over, a first point, and at least one referenceelectrode at, or in proximity to, a second point, wherein the firstpoint is located at the central nerve system of a vertebrate being, andthe second point is located outside the central nervous system of thevertebrate being, such as at the abdomen; and enhancing a neuralconnection between the first point and the second point by passing anelectrical current between the at least one active electrode and the atleast one reference electrode. In one embodiment, the referenceelectrode is a split electrode.

Trans-spinal direct current (tsDC) may be used to provide stimulation tothe damaged or dysfunctional portions of the central nervous systemand/or peripheral nerves. Trans-cranial DC stimulation (tcDC) may beused to modulate the excitability of the motor cortex, ameliorate theperception of pain, modulate cognitive functions, and/or treatdepression. These modalities may be used alone or in conjunction witheach other.

In one embodiment, the first point can be located at the spinal columnof the vertebrate being. According to this embodiment, the electricalcurrent can be provided by a stimulator including at least onestimulator unit that applies electrical pulses across the at least oneactive electrode and the at least one reference electrode. One or moreadditional reference electrodes can be placed at, or in proximity to, anadditional point located outside the central nervous system of thevertebrate being. If the vertebrate being is a human, the second pointand the additional point can be located at the pelvis of the human.

In another embodiment, an additional electrical stimulus can be appliedto the motor cortex of the vertebrate being during passing of theelectrical current between the first and second point (or “the primaryelectrical current”) The additional electrical stimulus can be a localstimulus in the form of at least one electrical pulse. The at least oneelectrical pulse can be applied synchronously with, or asynchronouslyfrom, the passing of the primary electrical current.

In even another embodiment, an additional electrical stimulus can beapplied to at least one muscle of the vertebrate being during passing ofthe primary electrical current. The additional electrical stimulus canbe a stimulus in the form of at least one electrical pulse. The at leastone electrical pulse can be applied synchronously with, orasynchronously from, the passing of the primary electrical current.

In yet another embodiment, a first additional electrical stimulus can beapplied to the motor cortex of the vertebrate being during passing ofthe primary electrical current, and a second additional electricalstimulus can be applied to at least one muscle of the vertebrate beingduring passing of the primary electrical current. The first additionalelectrical stimulus can be a local stimulus in the form of at least onefirst electrical pulse, and the second additional electrical stimuluscan be a stimulus in the form of at least one second electrical pulse.The at least one first electrical pulse and the at least one secondelectrical pulse can be applied synchronously with, or asynchronouslyfrom, the passing of the primary electrical current.

In still another embodiment, the primary electrical current is passed asa plurality of pulses, wherein each of the plurality of pulses has aduration from 0.5 ms to 5 ms.

In a further embodiment, the primary electrical current is passed aplurality of pulses having a frequency from 0.5 Hz to 5 Hz.

In an even further embodiment, a prompt to move a limb can be applied tothe vertebrate being during, or immediately before, the passing of theprimary electrical current. The prompt can be an aural prompt, a visualprompt, or a tactile prompt. The vertebrate being can be a human, andthe prompt can be provided by another human to the human. Alternativelyor additionally, the prompt can be provided by an automated control unitconfigured to generate the prompt in synchronization with the passing ofthe primary electrical current.

In a yet further embodiment, the vertebrate being can be a mammal, andsecond points can be located at a muscle in a limb of the mammal.

In a still further embodiment, the vertebrate being can be a human, andsecond points can be located at a muscle in a human limb.

According to another aspect of the present disclosure, another method ofimproving a neuromuscular condition of a vertebrate being is provided.The method includes: placing at least one active electrode at, or inproximity to, a first point located on one side of a spinal column of avertebrate being and at least one reference electrode at, or inproximity to, a second point, wherein the first point is located on theopposite side of the spinal column, wherein locations of the first pointand the second point are independently selected from the motor cortexand a muscle of the vertebrate being; and enhancing a neural connectionbetween the first point and the second point by passing an electricalcurrent between the at least one active electrode and the at least onereference electrode (or “the primary electrical current”). At least onepath of the primary electrical current can run across the spinal columnand between the first point and the second point.

In one embodiment, the at least one path of the primary electricalcurrent includes a motor pathway between the motor cortex and a muscle.The first point can be a point at the motor cortex and the second pointcan be a point at a muscle. Alternatively, the second point can be apoint at the motor cortex and the first point can be a point at amuscle.

In another embodiment, the first point is a point at a first muscle, andthe second point is a point at a second muscle that is different fromthe first muscle and located on the opposite side of the spinal columnfrom the first muscle. The at least one path of the primary electricalcurrent includes at least one first lower motoneuron connected to thefirst point and at least one second lower motoneuron connected to thesecond point.

In even another embodiment, the at least one active electrode is asingle active electrode, and the at least one reference electrode is asingle reference electrode.

In yet another embodiment, the at least one active electrode is aplurality of active electrodes or the at least one reference electrodeis a plurality of reference electrodes.

In still another embodiment, each of the at least one active electrodeand the at least one reference electrode can be attached to the motorcortex or a muscle of the vertebrate being topically, underneath a skin,or by surgical implantation.

In still yet another embodiment, the method can further includeidentifying a motoneuron that affects movement of a muscle of thevertebrate being in the spinal column, wherein the muscle issubsequently attached to one of the at least one active electrode or theat least one reference electrode. The method can further include:determining a maximal stimulus strength for the motoneuron at which nofurther increase in muscle contraction of the muscle is observed with anincrease in strength of electrical stimulation to the motoneuron; andsetting a voltage differential between at least one active electrode andthe at least one electrode during the passing of the current inproportion to the determined maximal stimulus strength. The voltagedifferential can be set at a same voltage as the maximal stimulusstrength.

In a further embodiment, the primary electrical current can be passed asa plurality of pulses, wherein each of the plurality of pulses has aduration from 0.5 ms to 5 ms.

In an even further embodiment, the primary electrical current can bepassed a plurality of pulses having a frequency from 0.5 Hz to 5 Hz.

In a yet further embodiment, the principle of the dipolarcortico-muscular stimulation can be achieved by applying localelectrical stimulation on the target muscle at the same time of askingthe individual to attempt to move the limb. As a person tries toactivate the nerve muscle, a signal from the brain and through thespinal cord would be in its way to the group of muscles under activity.If the two signals—a first signal from the brain and a second signalfrom the nerve by electrical stimulation—meet at the level of spinalcircuits, the connection specific to this group of muscles will bemarkedly strengthened. This principle has been applied to patients withneurologic pathology (e.g. cerebral palsy) and significant improvementshave been demonstrated.

Accordingly, the method can further include providing a prompt to move alimb to the vertebrate being during, or immediately before, the passingof the primary electrical current. The prompt can be an aural prompt, avisual prompt, or a tactile prompt. The vertebrate being can be a human,and the prompt can be provided by another human to the human. The promptcan be provided by an automated control unit configured to generate theprompt in synchronization with the passing of the primary electricalcurrent.

The vertebrate being can be a mammal, and one of the first and secondpoints can be located at a muscle in a limb of the mammal. Thevertebrate being can be a human, and one of the first and second pointscan be located at a muscle in a human limb.

In a still further embodiment, the primary electrical current can beprovided by a stimulator that applies a first voltage to the at leastone active electrode and a second voltage to the at least one referenceelectrode simultaneously. The primary electrical current can flowthrough a plurality of paths, and the plurality of paths can include afirst path between the motor cortex and a muscle and a second pathbetween two different muscles. Each of the plurality of paths can runacross the spinal column, wherein at least one path of the primaryelectrical current runs across the spinal column.

In still yet further embodiment, one or more additional electricalstimuli can be employed synchronously or asynchronously with the flowingof the primary electrical current. The stimulator may include aplurality of stimulator units so that the additional stimulus/stimulican be provided by a different stimulator unit than the stimulatorunit(s) that provide(s) the primary electrical current between the firstpoint and the second point. The additional stimulus/stimuli can beapplied between a pair of muscles so that the additional electricalcurrent flows through a different path that crosses the spinal column.

According to yet another aspect of the present disclosure, a system forimproving a neuromuscular condition of a vertebrate being is provided.The system includes: at least one active electrode, each sized andconfigured to be placed at, or in proximity to, a first point located atthe central nerve system of a vertebrate being; at least one referenceelectrode, each sized and configured to be placed at, or in proximityto, a second point that is located outside the central nervous system ofthe vertebrate being; a stimulator unit configured to generateelectrical stimulation waveforms; and at least one first lead wire thatcouples the stimulator unit to the at least one active electrode and atleast one second lead wire that couples the stimulator unit to the atleast one reference electrode, wherein the system is configured to forma current path through a motor pathway across the spinal column betweenthe first point and the second point.

In one embodiment, each of the at least one active electrode can besized and configured to be placed at, or in proximity to, a spinalcolumn of the vertebrate being. The at least one active electrode andthe at least one reference electrode can be sized and configured to beplaced at, or in proximity to, the spinal column of a human.

In another embodiment, the system can further include at least anotheractive electrode and at least another reference electrode, each sizedand configured to be placed at, or in proximity to, the motor cortex ofthe vertebrate being. The at least another active electrode and the atleast another reference electrode can be sized and configured to beplaced at, or in proximity to, the motor cortex of a human. The systemcan further include another stimulator unit configured to generateadditional electrical stimulation waveforms that are applied across theat least another active electrode and the at least another referenceelectrode. The stimulator unit and the other stimulator unit can besynchronized to provide the electrical stimulation and the additionalelectrical stimulation simultaneously.

In yet another embodiment, the system can further include at leastanother active electrode and at least another reference electrode, eachsized and configured to be placed at, or in proximity to, a muscle ofthe vertebrate being. The at least another active electrode and the atleast another reference electrode can be sized and configured to beplaced at, or in proximity to, a muscle of a human. The stimulator unitand the other stimulator unit can be synchronized to provide theelectrical stimulation and the additional electrical stimulationsimultaneously.

In still another embodiment, the system can further include prompt meansfor providing a prompt to move a limb to the vertebrate being during, orimmediately before, the passing of the electrical current.

According to still another aspect of the present disclosure, anothersystem for improving a neuromuscular condition of a vertebrate being isprovided. This system includes: at least one active electrode, eachsized and configured to be placed on, or in proximity to, a first pointthat is selected from the motor cortex and a muscle and is located onone side of a spinal column of a vertebrate being; at least onereference electrode, each sized and configured to be placed on, or inproximity to, a second point that is selected from the motor cortex anda muscle and is located on the opposite side of the spinal column; astimulator unit configured to generate electrical stimulation waveforms;and at least one first lead wire that couples the stimulator unit to theat least one active electrode and at least one second lead wire thatcouples the stimulator unit to the at least one reference electrode,wherein the system is configured to form a current path through a motorpathway across the spinal column between the first point and the secondpoint.

In one embodiment, one of the at least one active electrode and the atleast one reference electrode is sized and configured to be placed on,or in proximity to, the motor cortex. This electrode can be sized andconfigured to be placed on, or in proximity to, the motor cortex of amammal having limbs. Alternatively or additionally, this electrode oranother electrode can be sized and configured to be placed on, or inproximity to, the motor cortex of a human.

In another embodiment, all of the at least one active electrode and theat least one reference electrode are sized and configured to be placedon, or in proximity to, a muscle of the vertebrate being. All of the atleast one active electrode and the at least one reference electrode canbe sized and configured to be placed on, or in proximity to, a muscle ina limb of a mammal having limbs. Further, all of the at least one activeelectrode and the at least one reference electrode can be sized andconfigured to be placed on, or in proximity to, a human limb.

In even another embodiment, the at least one active electrode is asingle active electrode, and the at least one reference electrode is asingle reference electrode.

In yet another embodiment, the at least one active electrode is aplurality of active electrodes or the at least one reference electrodeis a plurality of reference electrodes.

In still another embodiment, each of the at least one active electrodeand the at least one reference electrode is configured to be attached tothe motor cortex or a muscle of the vertebrate being topically,underneath a skin, or by surgical implantation.

In still yet another embodiment, the system further includes at leastone probe for identifying a motoneuron that affects movement of a muscleof the vertebrate being and located in the spinal column by applyingelectrical voltage thereto.

In a further embodiment, the stimulator is configured to pass theelectrical current as a plurality of pulses having a duration from 0.5ms to 5 ms.

In an even further embodiment, the stimulator is configured to pass theelectrical current as a plurality of pulses having a frequency from 0.5Hz to 5 Hz.

In a yet further embodiment, the system further includes prompt meansfor providing a prompt to move a limb to the vertebrate being during, orimmediately before, the passing of the electrical current. The promptcan be an aural prompt, a visual prompt, or a tactile prompt. The promptmeans can be an automated control unit configured to generate the promptin synchronization with the passing of the electrical current.

In a still further embodiment, the stimulator is configured to apply afirst voltage to the at least one active electrode and a second voltageto the at least one reference electrode simultaneously.

In further another embodiment, the stimulator is configured to pass theelectrical current flows through a plurality of paths. The plurality ofpaths can include a first path between the motor cortex and one of theplurality of muscles and a second path between two of the plurality ofmuscles.

In even further another embodiment, the stimulator can be configured toapply a first voltage to the at least one active electrode and a secondvoltage to the at least one reference electrode simultaneously. Theelectrical current can be provided by the stimulator including at leastone stimulator unit that applies the first voltage to the at least oneactive electrode and the second voltage to the at least one referenceelectrode to improve the neuromuscular condition of the vertebratebeing. The at least one stimulator unit can apply the first voltage andthe second voltage simultaneously to improve the neuromuscular conditionof the vertebrate being.

In yet further another embodiment, the at least one stimulator unit caninclude a plurality of stimulator units. The first voltage can beapplied by a first stimulator unit and the second voltage can be appliedby a second stimulator unit simultaneously. Further, polarizing currentcan be delivered between the brain of the vertebrate being and a muscleof the vertebrate being by employing a third stimulator unit to improvethe neuromuscular condition of the vertebrate being. The thirdstimulator unit can be synchronized with the first and second stimulatorunits so that the polarizing current is delivered simultaneously withthe first voltage and the second voltages. Alternatively, the thirdstimulator unit can be operated independently from the first and/orsecond stimulator unit(s) so that the polarizing current is deliveredasynchronously from the first voltage and/or the second voltage.

In still further another embodiment, the at least one stimulator unitcan be a plurality of stimulator units including a stimulator unitconfigured to apply the first voltage and the second voltagesimultaneously. Polarizing current can be delivered between the brain ofthe vertebrate being and a muscle of the vertebrate being employinganother stimulator unit such as the third stimulator unit. The otherstimulator unit, e.g., the third stimulator unit, can be synchronizedwith the stimulator unit that delivers the first voltage and/or thesecond voltage so that the polarizing current is deliveredsimultaneously with the first voltage and the second voltages.Alternatively, the other stimulator unit can be operated independentlyfrom the stimulator unit so that the polarizing current is deliveredasynchronously from the first voltage and the second voltages to improvethe neuromuscular condition of the vertebrate being.

The present disclosure provides new motor pathway therapies, whichprovide permanent restoration of neural communications after a series oftherapy sessions. The absence of the need for re-treatment is adistinguishing feature of the present disclosure over prior art methodssuch as functional electric stimulation (FES) that require periodicre-treatment of muscles. The motor pathway therapies of the presentdisclosure can repair neural connection to improve the function of anexisting communication pathway and/or can create/facilitate new neuronalgrowth for additional neural communication in such pathway. The motorpathway therapies of the present disclosure can permanently restorehealthy neural communications without the need for re-treatment in orderto continue to enjoy such improvement.

As an illustration of treatment of an partially or wholly immobilizedpatient, treatment of the inured neuronal motor pathway for severalshort stimulation sessions repeated over several weeks can result inpermanent neuronal pathway communication improvement: now the patientcan grasp an object. However, follow-on treatment to induce stillfurther restoration of neural health in such treated pathway would be anext step: now the treated patient can lift the grasped object.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of the basic configuration and setup forutilizing dipole cortico-muscular stimulation (dCMS).

FIG. 1B is an illustration of three phases of pulses designed toevaluate dCMS.

FIG. 2A is a photograph of a control animal showing the normal postureof the hind limbs.

FIG. 2B is a photograph of spinal cord cross-sectional slice taken fromthe thoracic level of a control animal, wherein WM is white matter andGM is gray matter.

FIG. 2C is a photograph of an animal with SCI showing the abnormalpattern of the hind limbs.

FIG. 2D is a photograph of a spinal cord cross-sectional slice takenfrom the thoracic level of an animal with SCI showing the lesionepicenter.

FIG. 2E is a graphical representation of a quantification of sparedwhite matter at the lesion epicenter of animals with SCI and controlanimals.

FIG. 3A illustrates the responses to the gastrocnemius muscle afterstimulation.

FIG. 3B is an illustration showing the identification of motoneuronswhen their spontaneous activity (upper panel) is time locked andspontaneous contractions at the ipsilateral muscle (lower panel).

FIG. 4A is an illustration of six superimposed spinal responses afterhomonymous gastrocnemius muscle stimulation.

FIG. 4B is an illustration of six superimposed spinal responses aftermotor cortex (M1) stimulation.

FIG. 4C is an illustration of six superimposed spinal responses afterdCMS.

FIG. 4D is a graphical representation of the average latency of spinalresponses after muscle stimulation, dCMS, and after M1 stimulation.

FIGS. 5A and 5B are graphical representations of contraction for thecontralateral muscle during dCMS in animals with SCI.

FIGS. 5C and 5D are graphical representations of contraction for theipsilateral muscle during dCMS in animals with SCI.

FIGS. 6A and 6B show a plot of contralateral gastrocnemius muscleactivity after dCMS (contaralateral) in animals with SCI.

FIGS. 6C and 6D show a plot of contralateral gastrocnemius muscleactivity after dCMS (contaralateral) in animals with SCI.

FIGS. 6E and 6F are graphical representations of muscle twitch forcebefore and after dCMS in animals with SCI (contralateral andipsilateral).

FIGS. 7A and 7B are graphical representations of muscle twitch forcebefore and after dCMS in control animals.

FIG. 8 is a graphical representation of a fidelity index analysis foranimals with SCI and control animals.

FIG. 9A shows a plot of spontaneous activity of spinal motoneuronsbefore and after dCMS intervention.

FIG. 9B is a graphical representation of firing rates during an entireexperiment for an animal with SCI.

FIG. 9C is a graphical representation of firing rates before and afterdCMS in control animals (contralateral and ipsilateral) and animals withSC (contralateral and ipsilateral).

FIG. 10 is a first configuration of a simulator and a plurality ofactive electrodes (labeled “+”) and a plurality of reference electrodes(labeled “−”).

FIG. 11 is a second configuration of a simulator including multiplesimulator units and electrodes attached thereto.

FIG. 12 is an exemplary setup employing the second configuration. Thissetup was also employed for an experimental setup for the studydescribed below.

FIG. 13 shows Hoechst stains of transverse spinal cord sections from asegment (˜1 cm in length) located directly under the stimulating tsDCelectrode. Spinal cord sections from mice that received stimulation(right) were similar to sections from unstimulated controls (left),showing no evidence of morphological changes.

FIGS. 14A-14F illustrate that changes caused by tsDC in the frequency,amplitude, and pattern of spontaneous activity recorded from the tibialnerve. FIGS. 14 A and 14B are examples of spontaneous activity recordedbefore (baseline), during, and after a-tsDC (A) or c-tsDC (B) are shown.

In FIG. 14C, the firing frequency during a-tsDC showed a significanteffect of condition (F=135.40, p<0.001, repeated measures ANOVA). Posthoc tests revealed a higher firing frequency during a-tsDC steps +1, +2,and +3 mA.

In FIG. 14D, firing frequency during c-tsDC also showed a significanteffect of condition (F=338.00, p<0.001, repeated measures ANOVA). Posthoc testing revealed a significant difference during c-tsDC steps −2,and −3 mA.

In FIG. 14E, spike amplitude during a-tsDC showed a significant effectof condition (H=738.14 p=0.001, Kruskal-Wallis ANOVA). Post hoc testsrevealed a higher spike amplitude during a-tsDC +2 and +3 mA.

In FIG. 14F, spike amplitude during c-tsDC also showed an effect ofcondition (H=262.40, p≦0.001, Kruskal-Wallis ANOVA). Post hoc testsrevealed a higher spike amplitude during c-tsDC. Error bars representS.E.M. *p<0.05 relative to baseline.

FIGS. 15A-15C show that cathodal stimulation may accessrhythm-generating circuitry in the spinal cord. In FIG. 5A,autocorrelogram of a-tsDC-induced activity shows no oscillation orbursting. In FIG. 5B, autocorrelogram of c-tsDC-induced activity showsstrong bursts by 10 ms and oscillations. In FIG. 5C, oscillatoryactivity was also induced by injecting the glycine and GABA receptorblockers picrotoxin and strychnine into the spinal cord at L3-L4.

FIGS. 16A-16C illustrate that a-tsDC and c-tsDC differently modulatedcortically-elicited TS twitches. In FIG. 16A, examples of TS twitchesevoked before (baseline), during, and immediately after a-tsDC areshown. Note that a-tsDC depressed the ability of the motor cortex toelicit TS twitches during stimulation, but facilitated twitches afterstimulation. In FIG. 16B, however, c-tsDC improved the ability of themotor cortex to elicit TS twitches during stimulation, but notafterwards. For each animal (n=5/group), the average of ten TS twitcheswas analyzed before stimulation (baseline), during the five intensitysteps, and after stimulation (0, 5, and 20 min) with a-tsDC asillustrated in FIG. 16C or c-tsDC as illustrated in FIG. 16D.

FIGS. 17A-17D demonstrate that tsDC induced changes incortically-elicited tibial nerve potentials. In FIG. 17A, latencies oftibial nerve potentials, measured from the stimulus artifact (SA) to thefirst deflection of the potential, were prolonged during a-tsDC andshortened after a-tsDC. Dashed vertical lines mark the points ofmeasurement. Note the difference in the scale bars. In FIG. 17B,latencies of cortically-elicited tibial nerve potentials were shortenedduring c-tsDC and prolonged afterwards. FIG. 17C illustrate that, fora-tsDC, there was a significant effect of condition (H=30.10, p<0.001,Kruskal-Wallis ANOVA). Post hocs revealed a significantly longer latencyduring +2 mA and a shorter latency afterwards. FIG. 17D illustrate that,for c-tsDC, there was also a significant effect of condition (H=29.84,p<0.001, Kruskal-Wallis ANOVA). Post hocs revealed a significantlyshorter latency during −2 mA and a longer latency afterwards. Error barsrepresent S.E.M. *p<0.05 relative to baseline.

FIGS. 18A-18D illustrate the effect of paired tsDC and repetitivecortical stimulation (rCES) on cortically-elicited TS twitches.Representative recordings of TS twitches before stimulation (baseline),during stimulation, and after stimulation are shown for a-tsDC (+2 mA)paired with rCES in FIG. 18A and c−tsDC (−2 mA) paired with rCES in FIG.18B. rCES was adjusted to give the maximal response (−5.5 mA) and wasdelivered at 1 Hz for 3 min. Both a-tsDC paired with rCES in FIG. 18Cand c-tsDC paired with rCES in FIG. 18D significantly improvedcortically-elicited TS twitches compared to baseline. Error barsrepresent S.E.M. *p<0.001 compared to baseline, Wilcoxon Signed RankTest

FIG. 19 is a hypothetical diagram illustrating possible changes inmembrane potential when the spinal electrode negative delivers apolarizing current (not to scale).

DETAILED DESCRIPTION OF THE INVENTION

As stated above, the present invention relates to a system and methodfor treating neuromuscular conditions through two-point electricalstimulation, which are now described in detail with accompanyingfigures. It is also noted that drawings are not necessarily drawn toscale.

As used herein, a “vertebrate being” refers to any biological animalthat has a spinal column, and includes humans and all animals classifiedunder subphyla Vertebrata.

As used herein, a “limb” is a leg, an arm, a wing, a flipper, a side ofa fin, or any anatomical equivalent thereof of a vertebrate being.

As used herein, a first element is placed “in proximity to” a secondelement if a predominant portion (greater than 50%) of electricalcurrent applied to the first element flows to the first element or viceversa. When multiple electrodes are said to be placed in proximity to apoint, a predominant portion of electrical current applied to themultiple electrodes flow through the point, and consequently through anelement including that point.

As used herein, a “point” refers to a tissue site of an animal or ahuman.

As used herein, an element is “configured to” perform an act if theelement is shaped, and includes all necessarily intrinsic features, toenable performance of the act as a natural consequence of having theshape and the necessary feature.

As used herein, an “active electrode” is an electrode to which anelectrical pulse is applied either as at least one positive voltagepulse or at least one negative voltage pulse. Therefore, an activeelectrode can be a positive electrode or a negative electrode dependingon the polarity of the applied electrical pulse.

As used herein, a “reference electrode” is an electrode that provides areference voltage to a vertebrate being while an active electrodeapplies an electrical pulse. A reference electrode may be held at aconstant electrostatic potential, or an electrical pulse may be appliedto the reference electrode such that the electrical pulse applied to thereference electrode has a polarity that is the opposite of the polarityof the electrical pulse applied to at least one active electrode. If atleast one active electrode is at least one positive electrode, areference electrode is a negative electrode, and vice versa.

As used herein, a “polarizing current” refers to a direct currentelectrical current that flows and through a neuron between a firstelectrode and a second electrode and causes polarization of electricalcharges in the neuron.

As used herein, a “central nervous system” is the set of a brain and aspinal column of a vertebrate being.

As used herein, a “lower motoneuron” or a “lower motor neuron” is amotor neuron connecting the spinal column to a muscle fiber(s) andincluding an axon that terminates at the muscle fiber(s).

The application of dipole cortico-muscular stimulation (dCMS) results ina remarkable enhancement of the excitability of the motor pathway. Thisenhancement was observed in both animals and humans. In control animalsand in SCI animals, which had severe locomotor impairment associatedwith signs of spastic syndrome, the effect was observed both in theipsilateral and contralateral pathways. Maximal threshold of theipsilateral cortex was reduced. Improvement in muscle strength wasaccompanied by an increase in spontaneous activity and potentiation ofevoked responses of the spinal motoneurons. Spinal motoneuronalresponses and muscle twitches evoked by stimulation of thecontralateral, non-treated M1 (motor cortex) were significantly enhancedas well. The dCMS-induced effect persisted beyond the phase ofstimulation and extended through the entire period of the experiment asexplained in detail further below.

The electrodes may be attached topically on the surface, or underneaththe skin, or surgically implanted. In one embodiment, an activeelectrode is situated on the motor cortex (first point) and a referenceelectrode is situated on the desired muscle (second point), allowing thecurrent to travel across the spinal cord. In another embodiment, anactive electrode is situated on the desired muscle (first point) and areference electrode is situated on the motor cortex (second point),again allowing the current to travel across the spinal cord. In yetanother embodiment, neither the active electrode nor the referenceelectrode is placed on the motor cortex. Instead, both the activeelectrode and reference electrode are placed on desired first and secondpoint muscles, which are on opposite sides of the body, allowing thecurrent to travel across the spinal cord.

In one embodiment of the present disclosure, a dipolar cortico-muscularstimulator can be employed to provide electrical pulses for the purposesof the present disclosure. FIG. 10 illustrates an exemplary connectionscheme employing a dipolar cortico-muscular stimulator. A dipolarcortico-muscular stimulator can include a stimulator box with a LCDDisplay or computer connections to a software control system. In anon-limiting illustrative example, a dipolar cortico-muscular stimulatorhaving the following configuration can be employed:

Pulse Type: Constant current

Wave form: Rectangular

Pulse duration 0.5 to 5 ms

Pulse amplitude 1 to 15 mA (Voltages at 1 to 35V)

Frequency range 0.5 to 5 Hz

Inherent Safety/shutdown features to prevent over stimulation

The outputs are connected in a way that makes the stimulus intensity tobe the difference between the voltages at the positive and negativeoutputs. The regulations of both outputs are synchronized to make theabsolute value of the difference between these two outputs always thesame. Thus, when the positive output increases the negative outputshould decrease the same amount. For example, when the positive outputis increased from +4 V to +5 V, the negative output decreases from −1 Vto 0 V.

Digital-to-analog converter (DAC) can be used to provide analog output,i.e., stimulation, through analog outputs of the stimulator box. TheDACs can produce constant DC voltage levels or waveforms under softwarecontrol. The output of the DACs may be fed through a programmableattenuation network to produce different output ranges. The signal maybe then split into a positive and negative output through bufferamplifiers.

Optionally, each of the electrode wires can be split and connected tomultiple locations. For example, active electrode can be split intomultiple wires each with its own electrode. This is important in humanapplication in case more areas needed to be stimulated. For example, atthe cortex, an operator can use only one active electrode for focalstimulation or two active electrodes for more broad but less painfulstimulation. Also, at the muscle, the operator can include more parts ofthe limb in the same session. Individual electrode size should be about5 cm².

This system can be employed to improve a neuromuscular condition of thevertebrate being. The at least one active electrode is placed at, or inproximity to, a first point. The at least one reference electrode isplaced at, or in proximity to, a second point. As discussed above, eachof the first point is located on one side of a spinal column of avertebrate being, and each of the second point is located on theopposite side of the spinal column. Each location of the first point andthe second point can be independently selected from the motor cortex anda muscle of the vertebrate being. Each muscle includes at least onenerve. Electrical current is passed between the at least one activeelectrode and the second electrode. At least one path of the electricalcurrent runs across the spinal column and between the first point andthe second point.

In one embodiment, one of the at least one active electrode and the atleast one reference electrode can be sized and configured to be placedat, or in proximity to, the motor cortex. Such an electrode can be sizedand configured to be placed at, or in proximity to, the motor cortex ofa mammal having limbs or the motor cortex of a human. The at least oneactive electrode and the at least one reference electrode can be placedon the vertebrate being such that the at least one path of theelectrical current includes a motor pathway between the motor cortex anda muscle. The first point can be a point at the motor cortex and one ofthe second point can be a point at a muscle. Alternatively, the secondpoint can be a point at the motor cortex and the first point can be apoint at a muscle.

In another embodiment, all of the at least one active electrode and theat least one reference electrode can be sized and configured to beplaced at, or in proximity to, a muscle of the vertebrate being. Thus,all of the at least one active electrode and the at least one referenceelectrode can be sized and configured to be placed at, or in proximityto, a muscle in a limb of a mammal having limbs or a human limb. The atleast one active electrode and the at least one reference electrode canbe placed on the vertebrate being such that the first point is a pointat a first muscle, and the second point is a point at a second muscle.The at least one path of the electrical current can include at least onefirst lower motoneuron connected to the first point and at least onesecond lower motoneuron connected to the second point.

The at least one active electrode can be a single active electrode, andthe at least one reference electrode can be a single reference electrodeas illustrated in FIG. 1A. Alternatively, the at least one activeelectrode can be a plurality of active electrodes and/or the at leastone reference electrode can be a plurality of reference electrodes asillustrated in FIGS. 10 and 11.

If multiple electrodes are employed for either the at least one activeelectrode or the at least one reference electrode, the multipleelectrodes can be placed at, or in proximity with, the same muscle. Forexample, a plurality of first electrodes can be placed at, or inproximity with, the motor cortex, and a plurality of second electrodescan be placed at, or in proximity with, a muscle. Further, a pluralityof first electrodes can be placed at, or in proximity with, a firstmuscle, and a plurality of second electrodes can be placed at, or inproximity with, a second muscle that is different from the first muscle.In each of the examples above, the at least one active electrode can bethe plurality of first electrodes and the at least one referenceelectrode can be the plurality of second electrodes, or vice versa.

Each of the at least one active electrode and the at least one referenceelectrode can be configured for attachment to the motor cortex or amuscle of the vertebrate being by any method, and particularly,topically, underneath a skin, and/or by surgical implantation. In thiscase, the method of the present disclosure can include attaching each ofthe at least one active electrode and the at least one referenceelectrode to the motor cortex or a muscle of the vertebrate beingtopically, underneath a skin, and/or by surgical implantation.

In still yet another embodiment, the system can include at least oneprobe for identifying a motoneuron that affects movement of a muscle ofthe vertebrate being and located in the spinal column by applyingelectrical voltage thereto. An example of such at least one probe is thepair of pure iridium microelectrodes illustrated in FIG. 1A and labeledas “Rec.” If provided, the at least one probe can be employed toidentifying a motoneuron that affects movement of a muscle of thevertebrate being in the spinal column. The muscle is subsequentlyattached to an active electrode or a reference electrode. The at leastone probe can be employed to determine a maximal stimulus strength forthe motoneuron at which no further increase in muscle contraction of themuscle is observed with an increase in strength of electricalstimulation to the motoneuron. Then, a voltage differential between atleast one active electrode and the at least one electrode during thepassing of the current can be set in proportion to the determinedmaximal stimulus strength. For example, the voltage differential can beset at a same voltage as the maximal stimulus strength, or can be apredefined percentage of the maximal stimulus strength (e.g., 25% to200%).

In one embodiment, the stimulator can be linked to EMG(electro-myograph, muscle activity monitor) monitor to adjust the level(e.g. 50%) of muscle contraction at which the treatment session will bedelivered. Similar monitor for vital signs (heart rate; blood pressure,breathing rate) can be added. Electrode gel can be used to prevent burnsdue to electrolysis.

Another method of employing a dipolar cortico-muscular stimulator isillustrated in FIG. 11. The stimulation system includes multipleindependent stimulator units that are integrated in a single system,either in one box or in a plurality of boxes with electrical connectionstherebetween. A first stimulator unit, labeled “polarizing,” delivers apolarizing current between a point on a spinal column and a pointlocated outside of the central nervous system. Optionally, a secondstimulator unit, labeled “brain,” can deliver current to the motorcortex either synchronously with the polarization current orasynchronously with the polarization current to reinforce thestimulation provided by the first stimulator. Optionally, a thirdstimulator unit, labeled “muscle 1,” can deliver current to a musclearea either synchronously with the polarization current orasynchronously with the polarization current to reinforce thestimulation provided by the first stimulator. The third stimulator unitcan be used with the second stimulator unit, or without the secondstimulator unit. Additional stimulator units, represented by a fourthstimulator unit labeled “muscle 2,” can be used with the thirdstimulator unit to deliver monopolar negative current to another musclearea.

The points at which the polarizing current is applied to a vertebratebeing are schematically illustrated in FIG. 12. While a mouse isschematically shown in FIG. 2, this configuration can be employed forany vertebrate being including a human. Specifically, an activeelectrode, labeled “tsDC,” is placed on a first point located at thespinal column, which can be at any level within the spinal columnbetween, and including, the first spinal cord level and the last spinalcord level. A reference electrode, labeled “Ref,” can be placed on asecond point located at any area other than the area of the centralnervous system, i.e., outside of the brain and the spinal column.Because simulation of an area of the spinal column contacted by theactive electrode is preferred than stimulation of the area contacted bythe reference electrode, the reference electrode is preferably placed atsome distance away from the spinal column. While the reference electrodeis shown as a single electrode in FIG. 12, the reference electrode canbe replaced with a plurality of reference electrodes as illustrated inFIG. 11. Using a plurality of reference electrodes instead of a singlereference electrode enhances the effect of the electrical stimulationprovided by the active stimulus because the current density at theplurality of reference electrodes can be maintained low, while thecurrent density at the active electrode can be maintained high.

Typically, the voltage at the reference electrode(s) is held constant,and the voltage at the active electrode has the form of electricalpulses with a pulse duration 0.5 to 5 ms and a frequency from 0.5 Hz to5 Hz, although lesser and greater pulse durations and lesser and greaterfrequencies can also be employed. The polarity of the electrical pulseapplied to the active electrode can be either positive or negativedepending on applications.

In case the vertebrate being is a human, a pair of reference electrodesplaced on an anterior pelvis can provide effective stimulation to anarea of the spinal column. One of the most effective configurations forplacement of a pair of reference electrodes employs a point at theanterior superior iliac spine on the right side and a point at theanterior superior iliac spine on the left side. In this case, a secondpoint for placing a reference electrode in an embodiment employing asingle reference electrode is replaced by a second point and anadditional point on which two reference electrodes are placed. In otherwords, a reference electrode for spinal polarizing current can beimplemented as a pair of reference electrodes that are split and placedover the right and left anterior superior iliac spines. The pair ofreference electrodes is held at the same electrostatic potential.

The location of the first point, i.e., the point at which the activeelectrode is placed, depends on the nature of the neuromuscularcondition for which the treatment is performed. The location of thefirst point can be selected to maximize the effect of the treatment. Forexample, if the treatment is intended to improve the neuromuscularcondition of a vertebrate being for injuries suffered at a location inthe spinal column, the first point can be located in a spinal cord levelimmediately above, i.e., immediately more proximal to the brain than,the site of the spinal injury. In other words, for treatment of a spinalcord injury, the active electrode of polarizing current can be placed sothat the primary current passes through the injury site. An activeelectrode is placed at the spinal cord level immediately above theinjury site, and reference electrodes can be placed as described above.In one embodiment, repetitive stimulations at the brain (pulsed DCcurrent that are applied synchronously with, or asynchronously from, theprimary electrical current through the active electrode and thereference electrode(s)) can be paired with the polarizing spinalcurrent.

If the treatment is intended to improve the neuromuscular condition of avertebrate being for conditions caused by a trauma or a dysfunction inthe brain, the first point can be located at the spinal cord level one,i.e., the part of the spinal column closest to the brain. Conditionscaused by a trauma or a dysfunction in the brain include suchdisabilities as cerebral palsy, amyotrophic lateral sclerosis (ALS,otherwise known as Lou Gehrig's disease), traumatic brain injury,stroke, etc. In other words, for treatment of conditions where theinjury is located in the brain, the polarizing electrode can be locatedon the spinal area innervating the target limb. For treatment ofconditions affecting lower extremities, the active polarizing electrodeshould be situated at vertebral level T10 to L1 above the lumbarenlargement. For treatment of conditions affecting upper extremities,the active polarizing electrode can be placed at the level of T2 andbelow. In one embodiment, repetitive stimulations at the brain (pulsedDC current that are applied synchronously with, or asynchronously from,the primary electrical current through the active electrode and thereference electrode(s)) can be paired with the polarizing spinalcurrent.

For treating a condition such as ALS, stimulation intervention can alsobe applied to target muscles (in the form of localized pulsed DCcurrent) affected by the condition, simultaneously with application ofthe polarizing current to a spinal cord region innervating the targetmuscles and application of local stimulation to the motor cortex (in theform of localized pulsed DC current). These treatments should berepeated at different areas according to the condition.

If the treatment is intended to improve the neuromuscular condition of avertebrate being for injuries to, or disabilities caused by amalfunction at, a peripheral nerve, the first point can be located in aspinal cold level at which a corresponding lower extremity circuit islocated, and preferably at a spinal cord level that is most proximal tothe location of the injury or the disability. Conditions caused by aninjury or a disability located at a nerve include, for example,peripheral palsy, Erb's palsy, and/or other peripheral nerve injuriesdue to nerve compression, tension, or torsion (e.g., sciatica). Fortreating a condition such as Erb's palsy, stimulation intervention canalso be applied to target muscles (in the form of localized pulsed DCcurrent) affected by the condition, simultaneously with application ofthe polarizing current to a spinal cord region innervating the targetmuscles and application of local stimulation to the motor cortex (in theform of localized pulsed DC current). These treatments should berepeated at different areas according to the condition.

The electrical simulation to the spinal column can be provided alone orin combination with additional electrical stimulations to the brainand/or to at least one muscle. The effectiveness of synchronous orasynchronous application of additional electrical simulation to thebrain and/or the at least one muscle depends on the nature of the injuryor disability.

An electrical stimulation to the brain is schematically illustrated inFIG. 12 by two electrodes placed at the motor cortex of a vertebratebeing. The electrical stimulation provided to the brain is a localstimulation in which an area of the motor cortex of the electrical beingis stimulated synchronously with, or asynchronously from, the electricalstimulation of the spinal column by the first stimulator unit. The localelectrical stimulation to the motor cortex can be applied employing aconcentric electrode pair as illustrated in FIG. 12, or can be employedby a set of electrodes, e.g., a third electrode and a fourth electrodethat are placed at two different points at the motor cortex. The thirdelectrode and the fourth electrode are schematically shown in FIG. 11 astwo electrodes connected to the second stimulator unit labeled “Brain.”

Additional electrical stimulation can be provided to at least onemuscle, i.e., a single muscle or a plurality of muscles, synchronouslywith, or asynchronously from, the electrical stimulation of the spinalcolumn by the first stimulator unit. If a local electrical stimulationto the brain is employed, the additional electrical stimulation the atleast one muscle can be applied synchronously with, or asynchronouslyfrom, the local electrical stimulation to the brain by the secondstimulator unit. The additional electrical stimulation can be providedby a third stimulator unit and/or additional stimulator unit(s), such asthe stimulator units labeled “Muscle 1” and “Muscle 2” in FIG. 11. Asingle pair of electrodes or multiple pairs of electrodes can beconnected to a stimulator unit that stimulates a muscle. FIG. 12schematically illustrates an exemplary placement scheme for theadditional electrodes in which the additional electrodes are placed on aforelimb of a mouse. In general, at least one pair of additionalelectrodes can be placed at one or multiple pairs of points on any partof the body excluding the central nervous system, and particularly atany limb.

Electrodes connected to each of the stimulator units in FIG. 11 can be asingle pair of electrodes or multiple pairs of electrodes. Each pair ofelectrodes includes an active electrode and a reference electrode.Further, each reference electrode can be replaced with a plurality ofreference electrodes to prevent concentration of current to a singlereference electrode and to enable increase in the current density at thepoint at which the corresponding active electrode is present.

The second stimulator unit can deliver monopolar positive current to themotor cortex either synchronously with the polarization current orasynchronously with the polarization current to reinforce thestimulation provided by the first stimulator. Further, the thirdstimulator unit, labeled “muscle 1,” can deliver monopolar negativecurrent to a muscle area either synchronously with the polarizationcurrent or asynchronously with the polarization current to reinforce thestimulation provided by the first stimulator. Selecting the polarity ofthe electrical stimulations so that the voltages applied to the motorcortex is in general positive and the voltages applied to the at leastone muscle is in general negative can enhance the effectiveness of thetreatment, especially when the electrical stimulations are appliedsynchronously.

As discussed above, the first and second monopolar stimulator units ofFIG. 11 can be synchronized to deliver pulses simultaneously. Each unitcan have its independent control panel. The third, polarizing stimulatorunit can have the options to be either synchronized with the first andsecond stimulators, or can function independently, i.e., asynchronouslyfrom the first and second stimulators. In addition, the number ofelectrodes per connection (splitting into more than one electrode, e.g.4) can be as in the previous design as described above. For someapplications, a dipolar cortico-muscular stimulator in thisconfiguration is more preferable for human intervention because thestimulator gives more flexibility in designing stimulation patterns, andcan be safer and less painful.

In general terms, the invention described herein can be practicedemploying a system for improving a neuromuscular condition of avertebrate being. The system includes at least one active electrode, atleast one reference electrode, a stimulator, and at least one first leadwire and at least one second lead wire, which are employed to form anelectrical circuit that includes a vertebrate being.

Each of the at least one active electrode can be sized and configured tobe placed at, or in proximity to, a first point. The first point isselected from the motor cortex and a muscle, and is located on one sideof a spinal column of the vertebrate being. The at least one activeelectrode can be a single active electrode as illustrated in FIG. 1A(See the section on experimental data for description of components inFIG. 1A), or can be a plurality of active electrodes as illustrated inFIG. 10, or include a active electrode attached to a stimulator unit(labeled “brain”) and at least another active electrode attached toanother stimulator unit (labeled “polarizing”) as illustrated in FIG.11.

Each of the at least one reference electrode can be sized and configuredto be placed at, or in proximity to, a second point. The second point isselected from the motor cortex and a muscle, and is located on theopposite side of the spinal column. The at least one reference electrodecan be a single reference electrode as illustrated in FIG. 1A, or can bea plurality of reference electrodes as illustrated in FIG. 10, orinclude a reference electrode attached to a stimulator unit (labeled“muscle”) and at least another reference electrode attached to anotherstimulator unit (labeled “polarizing”) as illustrated in FIG. 11.

The stimulator can be configured to generate electrical stimulationwaveforms. Each of the at least one first lead wire couples thestimulator to an active electrode among the at least one activeelectrode. Each of the at least one second lead wire couples thestimulator to one of the at least one reference electrode. In oneembodiment, the system can be configured to form a current path througha motor pathway across the spinal column between the first point and thesecond point. In another embodiment, the system can be configured toform a current path between a first point in the spinal column and asecond point outside the central nervous system.

The stimulator can configured to pass the electrical current as aplurality of pulses having a duration from 0.5 ms to 5 ms, althoughlesser and greater durations can also be employed. Further, thestimulator can configured to pass the electrical current as a pluralityof pulses having a frequency from 0.5 Hz to 5 Hz.

The system can further include prompt means for providing a prompt tomove a limb to the vertebrate being during, or immediately before, thepassing of the electrical current. The prompt can be provided in any ofthe embodiments described above. The prompt can be an aural prompt, avisual prompt, or a tactile prompt. The prompt means can be an automatedcontrol unit configured to generate the prompt in synchronization withthe passing of the electrical current. The prompt means can be used forany vertebrate being capable of understanding the prompt, or trained torecognize the prompt (for example, by conditional reflexes). In thiscase, a prompt to move a limb can be provided to the vertebrate beingduring, or immediately before, the passing of the electrical current.The prompt can be provided by an automated control unit configured togenerate the prompt in synchronization with the passing of theelectrical current.

Alternatively or in addition, the vertebrate being can be a human, andthe prompt can be provided by another human to the human or to anon-human vertebrate being capable of understanding the prompt, ortrained to recognize the prompt. The other human can be a therapist. Inaddition, the prompt means can provide the prompt indirectly to thevertebrate being by first providing a direct prompt to the therapist ora trainer as the case may be, and then allowing the therapist or thetrainer to provide a prompt to the vertebrate being.

The vertebrate being can be a mammal, and the muscle can be a muscle ina limb of the mammal. The vertebrate being can a human, and the musclecan be a muscle in a human limb.

The stimulator can be configured to apply a first voltage to the atleast one active electrode and a second voltage to the at least onereference electrode simultaneously. Further, the stimulator can beconfigured to pass the electrical current flows through a plurality ofpaths as illustrated in FIGS. 10 and 11. The plurality of paths caninclude a first path between the motor cortex and one of the pluralityof muscles (for example, as provided by the first stimulator unit andthe second stimulator unit in FIG. 1 and a second path between two ofthe plurality of muscles (for example, as provided by the thirdstimulator unit). Each of the plurality of paths can run across thespinal column. In this case, at least one of the plurality of paths runacross the spinal column.

In the system of the present disclosure, the stimulator can beconfigured to apply a first voltage to the at least one active electrodeand a second voltage to the at least one reference electrodesimultaneously. Further, the stimulator can include at least onestimulator unit configured to provide the electrical current by applyinga first voltage to the at least one active electrode and a secondvoltage to the at least one reference electrode. In this case, theelectrical current can be provided by the stimulator including at leastone stimulator unit that applies the first voltage to the at least oneactive electrode and the second voltage to the at least one referenceelectrode to improve the neuromuscular condition of the vertebratebeing.

The at least one stimulator unit can be configured to apply the firstvoltage and the second voltage simultaneously. In this case, the atleast one stimulator unit can apply the first voltage and the secondvoltage simultaneously to improve the neuromuscular condition of thevertebrate being.

The at least one stimulator unit can include a plurality of stimulatorunits. A first stimulator unit can be configured to apply the firstvoltage and a second stimulator unit can be configured to apply thesecond voltage simultaneously with application of the first voltage bythe first stimulator unit. Thus, the first voltage can be applied by afirst stimulator unit and the second voltage can be applied by a secondstimulator unit simultaneously.

The plurality of stimulator units can further include a third stimulatorunit configured to deliver polarizing current between the brain of thevertebrate being and a muscle of the vertebrate. Polarizing current canbe delivered between the brain of the vertebrate being and a muscle ofthe vertebrate being employing the third stimulator unit to improve theneuromuscular condition of the vertebrate being. The third stimulatorunit can be synchronized with the first and second stimulator units sothat the polarizing current is delivered simultaneously with the firstvoltage and the second voltages. Alternatively, the third stimulatorunit can be configured to operate independently from the first andsecond stimulator units so that the polarizing current is deliveredasynchronously from the first voltage and the second voltages. In thiscase, the third stimulator unit can be operated independently from thefirst and second stimulator units so that the polarizing current isdelivered asynchronously from the first voltage and the second voltages.

The at least one stimulator unit can be a plurality of stimulator unitsincluding a stimulator unit configured to apply the first voltage andthe second voltage simultaneously. The first voltage and the secondvoltage can be applied by a stimulator unit simultaneously. Anotherstimulator unit, such as the third stimulator unit, can be configured todeliver polarizing current between the brain of the vertebrate being anda muscle of the vertebrate being. In this case, polarizing current canbe delivered between the brain of the vertebrate being and a muscle ofthe vertebrate being employing another stimulator unit. The otherstimulator unit, e.g., the third stimulator unit, can be synchronizedwith the stimulator unit that delivers the first voltage and/or thesecond voltage so that the polarizing current is deliveredsimultaneously with the first voltage and the second voltages.Alternatively, the other stimulator unit can be configured to beoperated independently from the stimulator unit so that the polarizingcurrent is delivered asynchronously from the first voltage and thesecond voltages. In this case, the other stimulator unit is operatedindependently from the stimulator unit so that the polarizing current isdelivered asynchronously from the first voltage and the second voltagesto improve the neuromuscular condition of the vertebrate being.

In general, direct current (DC) stimulation is a non-invasive techniqueused to modulate the excitability of the central nervous system. When DCstimulation is delivered trans-cranially, a positively- ornegatively-charged stimulating electrode (anode or cathode,respectively) is positioned at the cortical area to be stimulated, whilea reference electrode is usually situated at a distance. Trans-cranialDC stimulation (tcDC) is used to modulate the excitability of the motorcortex, ameliorate the perception of pain, modulate cognitive functions,and/or treat depression. The effect of DC stimulation depends on thetopography of neurons relative to the applied field, interactionsbetween functional neuronal circuits, and the polarity of the electrode.For example, while cathodal stimulation depresses neuronal activity,anodal stimulation activates neurons.

The spinal cord contains various populations of excitatory andinhibitory interneurons that mediate cortical and sub-cortical inputs.By acting on these interneurons, as well as motoneurons and ascendingand descending processes, DC stimulation at the spinal level could exertmodulatory effects on cortical and sub-cortical inputs to the spinalcord. Although DC stimulation has been found to improve functionalrecovery after spinal cord injury, only a few studies have investigatedthe effects of trans-spinal direct current (tsDC) on the excitability ofspinal neurons, and its effects on corticomotoneuronal transmission havenever been investigated.

Research leading to the present disclosure show differential modulatoryeffects of tsDC polarity on spontaneous activity, which are shown below.Cortically-elicited triceps surae (TS) twitches were increased duringcathodal trans-spinal direct current (c-tsDC), then depressed aftertermination, and were decreased during anodal trans-spinal directcurrent (a-tsDC), then potentiated after termination. While a-tsDC andrCES produced similar effects as a-tsDC alone, c-tsDC and rCES showedthe greatest improvement in cortically-elicited TS twitches.

In one embodiment. DC stimulation can be employed to improve spinalresponses to cortical stimulation. In many neurological disorders,connectivity between the cortex and spinal cord is compromised (e.g.,spinal cord injury or stroke). Stimulation protocols can be employed tostrengthen spinal responses. As illustrated in the studies describedbelow, neuronal activity is important in shaping c-tsDC after-effects.Specifically, c-tsDC can optimize cortico-spinal activity duringstimulation, and depress it at other times. The ability of c-tsDC tointeract with conical activity to cause different outcomes is aninteresting phenomenon that can support many clinical uses of c-tsDC.Translating this to rehabilitative strategies, either artificialcortical stimulation (when voluntarily muscle activation is impossible)or voluntary training during the application of c-tsDC can be employedto strengthen signal responses. Moreover, the depressive effect ofc-tsDC can be used to manage spasticity resulting from many neurologicdisorders.

C-tsDC can cause motoneurons to be more responsive to synapticactivation, but less inclined to generate spontaneous activity. This mayexplain why cortically-elicited TS twitches were potentiated duringc-tsDC application. Moreover, pre-synaptic hyperpolarization has beenshown to increase excitatory post-synaptic potentials (EPSPs). SeeEccles J., Kostyuk, P. G., Schmidt, R. F., The effect of electricpolarization of the spinal cord on central afferent fibres and on theirexcitatory synaptic action, J. Physiol. 162: 138-150 (1962); Hubbard J.I. and Willis W. D., Hyperpolarization of mammalian motor nerveterminals, J. Physiol. 163: 115-137 (1962); Hubbard J. I., and Willis W.D., Mobilization of transmitter by hyperpolarization, Nature 193:174-175 (1962). Such hyperpolarization is expected to occur incortico-spinal tract terminals and in spinal interneurons between thecortico-spinal tract and spinal motoneurons. Thus, nerve terminalhyperpolarization and dendrite depolarization induced by c-tsDC wouldcause potentiation of cortically-elicited TS twitches.

In a study leading to the present disclosure presented below,cortically-elicited TS twitches were depressed following c-tsDC andpotentiated following a-tsDC. DC stimulation of the brain has similarresults, as anodal stimulation increases while cathodal stimulationdecreases the excitability of the motor cortex in humans and in mice.Anodal-induced excitability appears to depend on membranedepolarization, while cathodal-induced depression depends on membranehyperpolarization. In addition, after-effects of both anodal andcathodal stimulation involve the N-methyl-D-aspartate (NMDA) glutamatereceptor.

Paining rCES with c-tsDC can not only prevent depression ofcortically-elicited TS twitches after c-tsDC termination, but remarkablyimprove twitches C-tsDC seems to induce a polarizing pattern as shown inFIG. 19, including pre-synaptic hyperpolarization and post-synapticdepolarization within the corticomotoneuronal pathway.

In theory, neuronal compartments in close proximity to the negativeelectrode should depolarize, and distant compartments shouldhyperpolarize. Therefore, excitability of neurons with dendritesoriented dorsally and axons oriented ventrally should increase, andexcitability of neurons oriented in the opposite direction (ventral todorsal) should decrease. Reversing the direction of the polarizingcurrent should result in opposite changes of membrane potential. Thenegative (−) and positive (+) signs indicate the status of thetrans-membrane potential. CT, corticospinal tract; IN, interneuron; MN,motoneuron.

This pattern, combined with rCES, would evoke long-term potentiation.Specifically, pre-synaptic hyperpolarization has been shown to increasethe size of EPSPs, which would subsequently increase neurotransmitterrelease and thereby cortical input. Although a low frequency stimulationwas applied to the motor cortex in the study described below, the actualfrequency of conical input was probably much higher. In addition,post-synaptic depolarization would activate the NMDA receptor. Theassociation between pre-synaptic increase of neurotransmitter releaseand steady post-synaptic depolarization would trigger the induction oflong-term potentiation. This could serve as the main mechanism forc-tsDC-induced enhancement of cortically-elicited TS twitches.Furthermore, reduction of inhibitory inputs to spinal circuits couldalso mediate the after-effects of paired rCES and c-tsDC.

First Experiment

A new configuration of electrical stimulation is provided herein as itwas tested in anesthetized control and spinal cord injury (SCI) mice.Constant voltage output was delivered through two electrodes. While thenegative voltage output (ranging from −1.8 to −2.6V) was delivered tothe muscle (two-wire electrode, 500 μm), the positive output (rangingfrom +2.4 to +3.2V) was delivered to the primary motor cortex (M1)(electrode tip, 100 μm). The configuration was named dipolarcortico-muscular stimulation (dCMS) and consisted of 100 pulses (1 mspulse duration, 1 Hz frequency).

In experimental testing, constant voltage output was delivered throughtwo electrodes. While the negative voltage output (ranging from −1.8 to−2.6V) was delivered to the muscle, the positive output (ranging from+2.4 to 3.2V) was delivered to the primary motor cortex (M1). Theconfiguration consisted of 100 pulses (1 ms pulse duration, 1 Hzfrequency). In SCI animals, after dCMS, muscle contraction improvedremarkably at the contralateral (456%) as well as ipsilateral (457%)gastrocnemius muscle. The improvement persisted for the duration of theexperiment (60 min.). The enhancement of the muscle force wasaccompanied by the reduction of M1 maximal threshold and thepotentiation of spinal motoneuronal evoked responses at thecontralateral (313%) and ipsilateral (292%) sides of the spinal cord.Moreover, spontaneous activity recorded from single spinal motoneuronswas substantially increased contralaterally (121%) and ipsilaterally(54%). Interestingly, spinal motoneuronal responses and muscle twitchesevoked by stimulation of non-treated M1 (received no dCMS) weresignificantly enhanced as well. Similar results obtained from controlanimals albeit the changes were relatively smaller. These findingsdemonstrated that dCMS could improve functionality of motor pathway anddramatically attenuates the effects of spinal cord injury.

In SCI animals, after dCMS, muscle contraction improved markedly at thecontralateral (456%) and ipsilateral (457%) gastrocnemius muscles. Theimprovement persisted for the duration of the experiment (60 min). Theenhancement of the muscle force was accompanied by the reduction of M1maximal threshold and the potentiation of spinal motoneuronal evokedresponses at the contralateral (313%) and ipsilateral (292%) sides ofthe spinal cord. Moreover, spontaneous activity recorded from singlespinal motoneurons was substantially increased contralaterally (121%)and ipsilaterally (54%). Interestingly, spinal motoneuronal responsesand muscle twitches evoked by the test stimulation of non-treated M1(received no dCMS) were significantly enhanced as well. Similar resultsobtained from control animals albeit the changes were relativelysmaller. Conclusion. These findings demonstrated that dCMS could improvefunctionality of motor pathway and thus it may have therapeuticpotential.

Methods

Animals

Specifically, experiments were carried out on CD-1, male and femaleadult mice in accordance with National Institute of Health (“NIH”)guidelines. All protocols were approved by the College of Staten IslandIACUC. Animals were housed under a 12 h light-dark cycle with freeaccess to food and water.

Spinal Cord Contusion Injury

Mice were deeply anaesthetized with ketamine/xylazine (90/10 mg/kgi.p.). A spinal contusion lesion was produced (n=15 mice) at spinalsegment T13 using the MASCIS/NYU impactor. 1 mm-diameter impact head rod(5.6 g) was released from a distance of 6.25 mm onto T13 spinal cordlevel exposed by a T10 laminectomy. After injury, the overlying muscleand skin was sutured, and the animals were allowed to recover under a30° C. heating lamp. To prevent infection after the wound was sutured, alayer of ointment contained gentamicin sulfate was applied. Followingsurgery, animals were maintained under pre-operative conditions for 120days before testing. The time of recovery was selected to ensure thatanimals developed a stable chronic spinal cord injury.

Behavioral Testing

Behavioral testing (n=15 animals with SCI) was performed 120 dayspost-injury to confirm that animals developed behavioral signs oflocomotor abnormalities, spasticity syndrome, and sensorimotorincoordination at the hindlimbs. We have only used animals thatdemonstrated higher (proximately symmetrical in both hindlimbs)behavioral abnormalities. After acclimation to the test environment,three different testing procedures were used to quantify thesebehavioral problems.

Basso mouse scale (BMS): Motor ability of the hindlimbs was assessed bythe motor rating of Basso mouse scale (BMS). The following rating scalewas used: 0, no ankle movement; 1-2, slight or extensive ankle movement;3, planter placing or dorsal stepping; 4, occasional planter stepping;5, frequent or consistent planter stepping; no animal scored more than5. Each mouse was observed for 4 min in an open space, before a scorewas given.

Abnormal pattern scale (APS): After SCI, animals usually developedmuscle tone abnormalities that were exaggerated during locomotion andlifting the animal off the ground (by the tail). APS was developed toquantify the number of muscle tone abnormalities demonstrated by animalsafter SCI in two situations: on ground and off ground. The followingrating scale was used: 0, no abnormalities; 1, for each of the followingabnormalities: limb crossing of midline, abduction, and extension orflexion of the hip joint, paws curling or fanning, knee flexion orextension, ankle dorsi or planter flexion. The total score was the sumof abnormalities from both hindlimbs. The maximal score in APS was 12.Abnormal patterns were usually accompanied by spasmodic movements of thehindlimbs.

Horizontal ladder scale (HLS): For accurate placing for the hindlimb,animals had to have normal coordination between sensory and motorsystems. For testing sensorimotor coordination, a grid with equalspacing (2.5 cm) was used. Animals were placed on the grid and wereallowed to take 20 consecutive steps. Foot slips were counted as errors.

Electrophysiological Procedures.

Intact (n=10) and SCI (n=21) animals underwent a terminalelectrophysiological experiment. Animals were anesthetized usingketamine/xylazine (90/10 mg/kg i.p.), which was found to reservecorticospinal evoked potential. Electrophysiological procedures started˜45 min after the first injection of anesthesia to perform theexperiments at intermediate to light levels of anesthesia, asrecommended by Zandieh and colleagues. See Zandieh S., Hopf R., Redl H.,Schlag M. G., The effect of ketamine/xylazine anesthesia on sensory andmotor evoked potentials in the rat. Spinal Cord, 41:16-22 (2003). Thiswas determined by the presence of front or hind limb withdrawal reflex.As needed, anesthesia was kept at this level using supplemental dosages(˜5% of the original dose).

The entire dorsal side of each animal was shaved. The skin covering thetwo hindlimbs, lumbar spine, and the skull was removed. The twogastrocnemii muscles (right and left) were carefully separated from thesurrounded tissue preserving blood supply and nerves. The tendon of eachof the muscles was threaded with a hook shaped 0-3 surgical silk, whichwas connected to the force transducers. Next, a laminectomy wasperformed in the 2nd, 3rd, and 4th lumbar vertebrae (below the lesion inanimals with SCI); the 13th rib was used as a bone land mark to identifythe level of spinal column. Since spinal cord levels are ˜3 levelsdisplaced upward relative to vertebral levels, the recording was assumedto be performed at spinal cord levels: 5th and 6th lumbar and 1stsacral. A craniotomy was made to expose the primary motor cortex (M1)(usually the right M1) of the hindlimb muscles located between 0 to −1mm from the Bregma and 0 to 1 mm from midline. The dura was left intact.The exposed motor cortical area was explored with a stimulatingelectrode to locate the motor point from which the strongest contractionof the contralateral gastrocnemius muscle was obtained using the weakeststimuli. In experiments aimed to test the effect of dCMS onnonstimulated motor pathway, two craniotomies were made over the rightand left hind limb areas of M1.

Both hind and fore limbs and the proximal end of the tail were rigidlyfixed to the base. Both knees were also fixed into the base to preventtransmitting any movement from stimulated muscles to the body and viceversa. Muscles were attached to force displacement transducers and themuscle length was adjusted to obtain the strongest twitch force (optimallength). The head was fixed in a custom made clamping system. The wholesetup was placed on an anti-vibration table. Animals were kept warmduring the experiment with radiant heat.

A stainless steel stimulating electrode (500 μm shaft diameter, 100 μmtip) was set on the exposed motor cortex. Paired stainless steelstimulating electrode (˜15 mm spacing; 550 μm diameter) was placed onthe belly of the gastrocnemius muscle. The same electrode was alternatedbetween left and right muscles according to experimental procedure.Electrodes were then connected to stimulator outputs. Extracellularrecordings were made with pure iridium microelectrodes (0.180 shaftdiameter; 1-2 μm tip; 5.0 MΩ). Two microelectrodes were inserted throughtwo small openings that were carefully made into the spinal dura matteron each half (right and left) of the spinal cord. The insertion was madeat approximately the same segmental level of the spinal cord. Referenceelectrodes were placed in the tissue slightly rostral to the recordingsites. The ground electrodes were connected to the flap of skin near theabdomen. Motorized micromanipulators were used to advance themicroelectrodes into the ventral horns. Extracellular activity waspassed through a standard head stage, amplified, filtered (bandpass, 100Hz to 5 KHz), digitized at 4 KHz, and stored in the computer for furtherprocessing. A power lab data acquisition system and LabChart 7 softwareby ADInstruments, Inc, CO, USA were used to acquire and analyze thedata.

Once a single motoneuron was isolated at the left and right side of thespinal cord, few antidromic pulses (range, −9 to −10 V) were applied tothe homonymous gastrocnemius muscle. As described by Porter, thepresence of antidromically-evoked response with a short latency (3.45ms) indicated that the recording electrode was placed in the vicinity ofthe neuron innervating stimulated muscle. See Porter R., Earlyfacilitation at corticomotoneuronal neuronal synapses. J. Physiol.207:733-745 (19700. These recordings were also used to calculate thelatency of ipsilateral and contralateral spinal responses to musclestimulation. A cortical pre-test stimulation of 10 pulses (anodalmonopolar) at maximal stimulus strength (usually +8 to +10V) was appliedto the primary motor cortex (M1). Maximal stimulus strength was definedas the strength of stimulation when no further increase in musclecontraction was observed. This was also used to calculate the maximalthreshold of M1 stimulation.

Next, dCMS was applied through two electrodes as shown in FIG. 1A. Thepositive and negative voltage outputs were connected to electrodessituated on the primary motor cortex (M1), and on the contralateralgastrocnemius muscle, respectively. Each of the two gastrocnemii muscleswas attached to a force transducer (not shown). Recording from singlemotoneuron (Rec) was performed simultaneously on each side of the spinalcord below the lesion. In FIG. 1A, IGM represents the ipsilateralgastrocnemius muscle, and CGM represents the contralateral gastrocnemiusmuscle.

Specifically, the negative output was connected to an electrode situatedon the gastrocnemius muscle and the positive electrode was at M1. Thevoltage strength and polarity were computer-controlled. The strength ofdCMS stimulation was adjusted so that contraction of the ipsilateralmuscle (to M1) was at maximal strength which was reached just before theappearance of tail contraction (visually observed). This level ofresponse was achieved by simultaneously applying a negative output(range, −2.8 to −1.8 V) to the muscle and positive output (range, +2.2to +3.2 V) to M1. At this maximal strength, dCMS was delivered (100pulses, 1 ms pulse duration, 1 Hz frequency), 15 to 20 seconds after thestimulating paradigm was ended, a post-test (with identical parametersas pre-test) stimuli were delivered to M1.

FIG. 1B shows the experimental design for the pulsing, range, duration,number of pulses, and frequency. The experimental procedure includedthree phases designed to stimulate the preparation and to evaluate itsreactions to dCMS. The force of muscle contraction and cortically-evokedspinal responses were evaluated before and after the application of dCMSin Pre-test and Post-test phases by application of ten monopolar pulses.The type of stimulation and location of the stimulation and recordingelectrodes was the same in these two phases. During dCMS phase thepreparation was stimulated by application of the positive and negativepulses to the motor cortex (M1) and contralateral gastrocnemius muscle(CGM) respectively. While the number of pulses delivered during Pre- andPost-test phases was the same (10), the number of pulses deliveredduring dCMS was 100. The duration (1 ms) and the frequency ofstimulation (1 Hz) were the same in all three phases of the experiment.The shape of the stimulating current at each phase is shown. There was acontinuous recording of ipsilateral and contralateral muscle twitchesand evoked and spontaneous spinal activity during the entire experiment.

Spontaneous activity was followed for 5 min, then the experiment wasended and animals were injected with a lethal overdose of anesthesia. Ina subgroup of animals, the maximal threshold of M1 was re-tested. Inaddition, in this subgroup, in order to determine the long lastingeffect of dCMS, the magnitude of cortically-evoked muscle twitches andspinal responses were retested every 20 min for 60 min after dCMS.

White Matter Staining

At the end of each experiment, animals were injected with a lethal doseof Ketamine. Two parts of the spinal column (including vertebrae andspinal cord) were dissected, one part (1.5 cm) included the lesionepicentre and another part (˜0.5 cm) included the recording area (toconfirm the electrodes location). Tissues were kept overnight (4° C.) in4% paraformaldehyde in 0.1 m PBS and cryoprotected in 20% sucrose in PBSat 4° C. for 24 h. The spinal column was freeze mounted and cut into 30μm sections and placed on poly-L-lysine-coated glass slides. The spinalcolumn part including the lesion epicentre was sequentially sectionedfrom rostral. Slides were numbered to identify their locations relativeto the lesion epicentre.

Four slides from each SCI animal (n=6) containing the lesion epicentreand two slides containing no signs of damaged spinal cord tissue fromabove and below the lesion were taken for luxol fast blue (Sigma)staining. The lesion epicentre was identified as the section containingthe least amount of Luxol fast blue. Sections from control animals (n=3)at spinal cord T13 level were stained with luxol fast blue. Sectionsfrom the recording area were stained with cresyl violet.

The amount of spared white matter was measured using Adobe Photoshop CS4by Adobe Systems, San Jose, Calif., USA. To assess the extent of thespinal cord damage, the spared white matter at the lesion epicentre wascompared with the white matter at spinal cord level T13 in controlanimals.

Data Analysis

To evaluate the latencies, the time was recorded from the start of thestimulus artifact to the onset of the first deflection of spinalresponse. Measurements were made with a cursor and a time meter onLabChart software. The amplitude of spinal responses was measured aspeak-to-peak. Analysis of muscle contractions were performed with peakanalysis software by ADInstruments. Inc, CO, USA, as the height oftwitch force measured relative to the baseline. Spike Histogram softwarewas used to discriminate and analyze extracellular motoneuronalactivity. All data were reported as group means±standard deviation (SD).Paired student's t-test was performed for before-after comparison or twosample student's t-test to compare two groups; statistical significanceat the 95% confidence level (p<0.05). To compare responses from bothsides of spinal cords recorded from control animals and from animalswith SCI, one way ANOVA was performed followed with Solm-Sidak post hocanalysis. Statistical analyses were performed using SigmaPlot (SPSS,Chicago, Ill.), Excel (Microsoft, Redwood, Calif.), and LabChartsoftware (ADInstruments, Inc, CO. USA).

Results

1. Behavioral Assessment.

A contusion lesion of the spinal cord resulted in the appearance ofsigns of spasticity syndrome such as crossing of both limbs and fanningof the paws (compare 2A and 2C). These postural changes were quantifiedusing the abnormal pattern scale (APS). APS showed substantial increasefor both on (APS_(on) 9.8±0.70) and off (APS_(off) 9.8±0.70) groundconditions. These postural abnormalities were also accompanied byreduction in Basso Mouse Scale (BMS) scores from 9 in control mouse to1.2±0.47 and 1.0±0.63 for right and left hindlimb in SCI mouse (n=15),respectively. In addition, the number of errors on a horizontal laddertest was close to maximum (20) for left (19.5±0.50) and right(18.83±1.16) hindlimb. Collectively, these results indicate that spinalcord injury procedure used in the current study was reliable in inducingbehavioral signs of the injury. This strengthens the interpretation ofour data.

2. Anatomical Assessment.

FIG. 2A is a photograph of a control animal showing the normal postureof the hindlimbs. FIGS. 2B and 2D show photographs of cross-sectionalslices from the thoracic spinal cord region and the lesion epicentretaken from normal and SCI animals, respectively. The lesion size wasproximally equal in all injured animals tested histologically (n=6). Arim of white matter was spared on the lateral and ventral side of thespinal cord. The area of spared white matter at the lesion epicentre(0.06±0.03 mm2) was significantly reduced 16 weeks after SCI compared tothe area of white matter at the same spinal level (0.15±0.06 mm2) incontrol animals (n=3) (p=0.04, t-test), FIG. 2E. On average, the totalcross-sectional area (white and gray matters) of the lesion epicenterwas 75±14% of the total cross-sectional area of the same spinal level incontrol animals.

3. Spinal Motor Neuron Identification.

Spinal motoneurons (or motor neurons) innervating the gastrocnemiusmuscle were at first identified by their large spontaneous spikes. Themotoneuronal spike was also accompanied by a distinctive and crisp soundrecorded with a loud speaker. Second criterion used to identify spinalmotoneurons was their response to the stimulation of the gastrocnemiusmuscle. Stimulating the gastrocnemius muscle produced a short latencyantidromically-generated response that was recorded from motor neuronsin the ipsilateral spinal cord. Simultaneously, the microelectrode onthe contralateral side of the spinal cord recorded a response that hadrelatively longer latency than the one picked up from the ipsilateralside. In FIG. 3A, three representative conditions were seen during theidentification of motoneurons. The two panels, far left and middle, showsimultaneous motoneuronal responses to stimulated gastrocnemius muscle.The far left panel shows the response of the motoneuron in theipsilateral side. The middle panel shows the response of the motoneuronin the contralateral side. The far right panel shows a situation whenthe motoneuron was not responding to the antidromic stimulation of thehomonymous gastrocnemius muscle. This confirmed that the unit was notinnervating the stimulated gastrocnemius muscle. Third, as depicted inFIG. 3 B the muscle twitches (lower panel) were correlated withmotoneuron activity (upper panel). This association between spontaneousspikes and muscle twitches was used to confirm the connection. FIG. 31shows typical spike generated by motoneuron. Finally, it washistologically confirmed that recording electrodes were localized in theventral horn of the spinal cord.

4. Latencies.

Stimulating the gastrocnemius muscle resulted in short and long latencyspinal responses recorded by microelectrodes placed in the ipsilateraland contralateral ventral horns of the spinal cord, respectively. FIG.4A shows superimposed traces of 6 antidromically-evoked responses, andthe line marks the spinal responses. While the average latency ofantidromically-evoked responses was 3.45±1.54 ms, the average latency ofthe contralateral responses (not shown) was longer (5.94±1.24 ms)indicating a transynaptic pathway. The difference between ipsilateraland contralateral spinal responses was statistically significant (n=15,p<0.001, t-test). Stimulating M1 resulted in ipsilateral andcontralateral spinal motoneuronal responses.

FIG. 4B shows six superimposed contralateral responses after M1stimulation. The ipsilateral response is not shown in FIG. 4A or 4B. Theaverage latency of ipsilateral and contralateral responses was16.09±1.02 ms and 22.98±1.96 ms, respectively. The difference in latencybetween ipsilateral and contralateral responses (6.9 ms) wasstatistically significant (n=15, p<0.001, t-test). The application ofdCMS resulted in successive spinal motoneuronal responses picked up fromthe contralateral (to M1) electrode.

FIG. 4C shows six superimposed recorded traces. In FIG. 4C, threedistinctive responses are seen, one with short latency (3.45±1.54 ms),the second with longer latency (6.02±1.72 ms), and a third with muchlonger latency (19.21±2.28 ms) (n=15). The latency of the ipsilateral(to M1) spinal motoneuronal responses (not shown) was 6.02±2.8 ms.

FIG. 4D summaries the average latencies collected during muscle. M1, anddCMS paradigms. Ipsilateral spinal response to M1 stimulation (Ip) wasfaster than the contralateral response (Co) (p<0.05). Muscle stimulationgenerated shorter response at ipsilateral motoneuron than the ones atthe contralateral side (p<0.05).

5. Changes in Muscle Contraction and Spinal Responses During DipolarCortico-Muscular Stimulation (dCMS).

The application of dCMS gradually increased the twitch peak forcerecorded from the gastrocnemii muscles and neuronal activity recordedfrom the spinal cord. Since the magnitude of these enhancements weresimilar in control and injured animals, only data obtained from SCIanimals (n=9) are presented. The increase in the force of thecontralateral muscle contraction is shown in FIGS. 5A and 5B.

FIG. 5A shows that initial and final muscle twitches demonstratedgreater twitch peak force at the end (final) than the beginning(initial) of dCMS on the contralateral muscle to stimulated M1. WhileFIG. 5A depicts representative recordings, the averaged results obtainedfrom all 9 SCI animals are shown in FIG. 5B. The increase from aninitial twitch peak force of 4.8±1.12 g to a final twitch peak force of6.1±0.71 g was statistically significant (percent change=25.0±3.8%,p=0.001, paired t-test). The twitch peak force of ipsilateral muscleincreased as well.

Representative recordings and averaged results are shown in FIGS. 5C and5D. FIG. 5C shows initial and final muscle twitches of the ipsilateralmuscle (to stimulated M1) during dCMS, which demonstrated an increase intwitch force in response to dCMS. FIG. 5D is a bar graph showingaverages (n=9) of initial and final twitch peak force of the ipsilateralmuscle. The final twitch force increased significantly from its initialvalue of 1.8±0.74 g (percent change=37.7±1.14%; p=0.001, paired t-test).

Similar results were obtained by comparing the first and the last spinalmotoneuronal responses of the 100 pulses of dCMS protocol. On average,the contralateral (to stimulated M1) spinal motoneuronal responsesshowed significant increase (percent change=49.75±16.9%, p=0.013, onesample t-test), as did the ipsilateral (to stimulated M1) spinalmotoneuronal responses (percent change=48.10±19.8%, p=0.04, one samplet-test). These findings suggest that physiological processes thatmediate stronger connections of the corticomotoneuronal pathway wereinitiated during dCMS application.

6. The Influence of dCMS Application on Muscle Twitches and NeuronalActivity in SCI Animals.

Cortically induced muscle twitches (measured as peak twitch force) wereexamined before and after dCMS in SCI animals. In all animals used inthese experiments, twitch force was remarkably increased after dCMS. Anexample of twitches of the contralateral (to stimulated M1) (FIG. 6A)and ipsilateral (to stimulated M1) (FIG. 6C) gastrocnemius musclesbefore (upper panels) and after (lower panel) dCMS are shown in FIGS. 6Aand 6C. The cortically induced spinal responses (measured aspeak-to-peak) were also examined, which also substantially increased.Examples of contralateral (FIG. 6B) and ipsilateral (FIG. 6D) spinalresponses are shown.

In FIG. 6E, the twitch peak force of the contralateral muscle showedsignificant increase (n=9; p<0.001) (average before=0.50±0.28 g vs.average after=2.01±0.80 g) after dCMS, as did the twitch peak force ofthe ipsilateral (to stimulated M1) muscle (average before=0.21±0.12 vs.average after=1.36±0.77, p<0.001, paired t-test). In FIG. 6F, spinalmotoneuronal responses (n=9) contralateral (to stimulated M1) showedsignificant increase after dCMS (average before=347.67±294.68 μV vs.average after=748.90±360.59 μV, p=0.027, paired t-test) (increased by313±197%), as did ipsilateral (to stimulated M1) spinal motoneuronalresponses (average before=307.13±267.27 μV vs. averageafter=630.52±369.57 μV, p=0.001, paired t-test) (increased by 292±150%).Data are shown as means±SD. These results show that dCMS greatlypotentiates the motor pathway in injured animals.

The maximal cortical threshold defined as the lowest electrical stimuluseliciting the strongest muscle twitch peak force was reduced from9.4±0.89 V to =5.7±0.95 V after dCMS application (n=4, p<0.001, t-test).The muscle twitch force and the magnitude of spinal motoneuronalresponses, evaluated 60 min after dCMS in 5 SCI animals, were stillsignificantly elevated on both sides (repeated measure ANOVA followedwith post hoc, p<0.001).

7. Effects of dCMS on the Nonstimulated Cortico-Muscular Pathway inAnimals with SCI.

The test stimulation of the other M1, contralateral to M1 where dCMS hasbeen applied, revealed an increase of the contraction force recordedfrom contralateral and ipsilateral gastrocnemii muscles. The increase incontralateral (percent change=182.8±87.18%), and ipsilateral muscles(percent change=174.8±136.91%) was statistically significant (n=6,p<0.05, t-test).

Contralateral spinal motoneuronal response was increased significantly(p=0.006, t-test) (average percent change=373.8±304.99%), as didipsilateral (average percent change=289.2±289.62%, p=0.025, t-test).These results indicate that even though dCMS was unilaterally applied,it affected the cortico-muscular pathway bilaterally.

8. The Influence of dCMS Application on Muscle Twitches and NeuronalActivity in Control Animals.

The application of dCMS across the cortico-muscular pathway in controlanimals (n=6) resulted in an increase in the contraction force producedby both gastrocnemii muscles. FIGS. 7A and 7B show twitch force andcortically evoked spinal responses after dipolar cortico-muscularstimulation (dCMS) in normal mice. FIG. 7A is a quantification ofresults from 6 control animals, which revealed significant increase incontralateral (CO) and ipsilateral (Ips) (to stimulated M1) muscletwitch force after dCMS. FIG. 7B shows contralateral (to stimulated M1)cortically evoked spinal responses, which significantly increased afterdCMS, as did ipsilateral responses. The twitch peak force of thecontralateral muscle increased from 1.62±1.0 g before to 5.12±1.67 afterdCMS application (percent change=250.75±129.35%, p=0.001, paired t-test.FIG. 7A). The twitch peak force of the muscle on the ipsilateral sideincreased as well, although the increase was less pronounced (from0.16±0.05 g to 0.39±0.08 g, before and after dCMS, respectively (percentchange=166.36±96.56%, p=0.001, paired t-test, FIG. 7A).

The amplitude of evoked responses recorded from spinal motoneurons wasalso enhanced by dCMS application. As depicted in FIG. 7B, the averageamplitude of these spikes recorded at the contralateral side increasedfrom 127.83±46.58 μV to 391.17±168.59 μV (percent change=168.83±152.00%,p=0.009, paired t-test). The increase at the ipsilateral side was evengreater (percent change=369.00±474.00%, 77.50±24.73 μV before versus267.00±86.12 μV after dCMS, p=0.007, paired t-test).

9. Comparison Between Control and SCI Animals.

The cortically-induced twitches of the contralateral muscle, recordedfrom control animals were stronger than twitches observed in SCI animalsregardless of whether they were recorded before (p=0.009, t-test), orafter (p=0.001, t-test) the dCMS procedure. The response of ipsilateralmuscles, however, was more complex. Before dCMS, SCI animals showedhigher ipsilateral twitch peak force than control animals, although thedifference was not statistically significant (p=0.39, t-test). Thisdifference was significantly enhanced after dCMS intervention (p=0.01,t-test).

Similarly, before dCMS, the cortically-induced responses recorded fromspinal motoneurons were higher in SCI animals at ipsilateral andcontralateral sides, although the difference did not reach statisticalsignificance (p=0.13, t-test). However, following dCMS, this differencewas increased and became statistically significant (p=0.009, t-test).

Next a relative measure was obtained, which was characterized as a“fidelity index”. Fidelity index (FI) is the normalized conicallyinduced spinal motoneuronal response to the corresponding muscle twitchpeak force (spinal response/muscle twitch ratio). Lower fidelity indexvalue indicates better association between spinal responses and theircorresponding muscle twitches. In other words, it means better abilityof a spinal response to induce muscle contraction. Therefore, changes inthis index may indicate changes in relation between spinal andperipheral excitability.

After dCMS, SCI animals showed overall significant group reduction in FI(F=3.3, p<0.033, ANOVA) (FIG. 8). In FIG. 8, Solm-Sidak post hoc testshowed reduction in FI in contralateral (average before=368.35±342.51vs. average after=246.15±112.24), however, the difference was notstatistically significant (p=0.46). The ipsilateral FI was significantlyreduced after dCMS (average before=704.59±625.7 vs. averageafter=247.95±156.27) (p=0.011). The effect of dCMS treatment was theopposite in control animals which demonstrated overall group increase inFI after this procedure (F=31.51, p<0.001, ANOVA). FI was significantlyincreased after dCMS (Solm-Sidak post hoc, p<0.001) in the ipsilateralside (average before=328.53±104.83 vs. average after 526.83±169.36).There was also a trend reflecting an increase in the contralateral side(average before=48.59±17.71 vs. average after =56.15±24.19), but was notstatistically significant (Solm-Sidak post hoc, p=0.89).

Comparing FI from control animals with FI from SCI animals showed astatistically significant lower index in the contralateral side ofcontrol animals (p<0.001, ANOVA, Solm-Sidak post hoc) both before andafter dCMS. These results indicate that an inexcitability problem existsat the level of peripheral nerve and muscle.

10. Increase in Spinal Motoneurons Spontaneous Activity Due to dCMS.

Comparing the firing rate of spontaneous activity before and after dCMSintervention demonstrated significant increase in both control and SCIanimals. In FIGS. 9A and 9B, a representative spontaneous activityrecording from an SCI animal is shown. In SCI animals, spontaneousactivity was significantly increased in the contralateral side of thespinal cord (average before=17.31±13.10 spikes/s vs. averageafter=32.13±14.73 spikes/s; p=0.001) (121.71±147.35%), as it did in theipsilateral side (average before=18.85±13.64 spikes/s vs. averageafter=26.93±17.25; p=0.008) (percent change=54.10±32.29%). In controlanimals, spontaneous activity was significantly increased in thecontralateral (to stimulated M1) side of the spinal cord (averagebefore=11.40±8 65 spikes/s vs. average after=20.53±11.82 spikes/s;p=0.006) (percent change=90.10±42.53%), as it did in the ipsilateralside (average before=11.63±5.34 spikes/s vs. average after=22.18±10.35spikes/s, p=0.01) (percent change=99.10±1.10%). One way ANOVA showed nosignificant difference between control and SCI animals in firing rate,although, SCI animals demonstrated higher firing rate.

11. Effects of One Point (Monopolar) Stimulation of Muscle or Cortex.

In order to determine that the effect was unique to dCMS, the influenceof monopolar stimulation (maximal stimulation for 100 pulses, 1 Hzfrequency) of either the muscle or the motor cortex on spinalmotoneuronal response and muscle twitch peak force was examined.

As expected, muscle stimulation resulted in significant reduction inmuscle twitch force (−20.28±7.02%, p<0.001, t-test) (n=5, 3 SCI and 2control). It also resulted in a significant reduction in spinalmotoneuronal responses evoked by the contralateral (to stimulatedmuscle) M1 test stimulation (average before=747.50±142.72 μV, vs.average after=503.14±74.78) (F=17.11, one way ANOVA, Solm-Sidak posthoc, p<0.001), however, no significant change was seen in responsesrecorded in the ipsilateral (to stimulated muscle) side of the spinalcord (average before 363.33±140.67 μV vs. average after=371.43±35.61,p=0.84).

In a separate group of animals (n=5, 3 SCI and 2 control), the effect ofthe monopolar stimulation paradigm applied only at the motor cortex oncontralateral muscle twitch peak force and spinal motoneuronal responsewas tested. Both, the muscle twitch and motoneuron response weresignificantly reduced by over 50% (−53.69±4.3%, p=0.001, t-test) andalmost 15% (−14.59±9.10%, p=0.003, t-test), respectively. These resultsindicate that one point muscle or cortical stimulation at maximalstrength results in fatigue of muscle twitch force and reduction inspinal responses.

In general, the results show remarkable enhancement of the excitabilityof the motor pathway induced by unilateral application of dCMS. Thisenhancement was observed in control animals and in SCI animals that hadsevere locomotor impairment associated with signs of spastic syndrome.The effect was observed both in the ipsilateral and contralateralpathways. Maximal threshold of the ipsilateral cortex has been reduced.Improvement in muscle strength was accompanied by an increase inspontaneous activity and potentiation of evoked responses of the spinalmotoneurons. Spinal motoneuronal responses and muscle twitches evoked bystimulation of the contralateral, non-treated M1 were significantlyenhanced as well. The dCMS-induced effect persisted beyond the phase ofstimulation and extended through the entire period of the experiment (60min).

Bilateral responses to cortical stimulation have been routinelyobserved. They can be mediated by interhemispheric connections,ipsilateral cortico-spinal connections (5-6% of the contralateralprojections), or commissural spinal neurons. As seen in FIGS. 17F and18B, ipsilateral responses to unilateral stimulation of motor cortexevoked larger responses in SCI animals compared to controls. Theseresults further support the idea that ipsilateral corticospinalprojections are more efficient in evoking muscle contraction after SCI.

The mechanism of dCMS-induced increase in the efficiency of the motorpathway is not clear and one can only speculate what processes have beenmodulated. It is obvious that the potentiation in muscle force duringdCMS is not like the potentiation seen after neuromuscular stimulation.See Luke R, Harris W, Bobet J, Sanelli L, Bennett D J, Tail MusclesBecome Slow but Fatigable in Chronic Sacral Spinal Rats With Spasticity,J. Neurophysiol. 95:1124-1133 (2006). While neuromuscular stimulationleads to a brief potentiation of muscle force followed by a steepreduction in force, dCMS leads to a gradually proceeding increase in theamplitude of cortically-elicited muscle contraction. Since theenhancement occurred at contra- and ipsilateral sides, the locus ofpotentiation is most likely either spinal or supraspinal. Theenhancement of cortically-elicited muscle contraction was accompanied bya reduction in maximal threshold to conical stimulation, an increase inspinal motoneuronal responses, and an increase in cortically-elicitedspinal motoneuronal responses. Therefore, one can assume thatimprovements occurred simultaneously at several functional levels of thecorticomotoneuronal pathway.

In view of the fact that the current employed in the stimulationparadigm was always positive at one end and negative at the other, thestimulation can be considered in part polarizing. In the past, theparadigm of polarizing current was used to study excitability ofdifferent parts of the nervous system. See Landau W. M., Bishop G. H.,Clare M. H., Analysis of the form and distribution of corticalpotentials under the influence of polarizing currents, J. Neurophysiol.27:788-813 (1964); Gorman A. L. F., Differential patterns of activationof the pyramidal system elicited by surface anodal and cathodal corticalstimulation, J. Neuro Physiol. 29:547-64 (1965); Terzoulo C. A., BullockT. H., Measurement of imposed voltage gradient adequate to modulateneuronal firing, Proc. Natl. Acad. Sci. USA, 42:687-694 (1956); BindmanL. J, Lippold O C. J., Redfeamn J. W. T., Long-lasting changes in thelevel of the electrical activity of the motor cortex produced bypolarizing currents, Nature 196:584-585 (1962). In these studies,polarizing current produced potential membrane changes in whichhyperpolarization occurs at cellular parts near the positive electrodeand depolarization occurs near the negative electrode. Complying withthis rule, for example, the situation of two polarizing electrodes onthe spinal cord (one on the ventral side and the other on the dorsalside) produced changes in membrane and spike potentials of primaryfibres from muscles. See Landau et al. supra.

The results of the above study suggest that the current is polarizingduring the brief, steady moment of pulse duration (1 ms). Given theelectrodes placement, in which negative at the muscle and positive atthe cortex, the cell body of conticospinal neurons is expected tohyperpolarize and their nerve terminals depolarize. Moreover, spinalmotoneurons expected to hyperpolarize at the cell body and dendrites,and depolarize at the neuromuscular junction.

According to cell topography relative to the applied electrical field,membrane potential changes are also expected to occur at interveninginterneurons. These membrane changes that occur briefly during eachpulse of dCMS, seem to prime corticomotoneuronal pathway forpotentiation. In addition, the stimulating pulse has two more periods:rising (0.250 ms) and falling (0.250 ms). These changing periods causeda flow of current that exited from one end and entered at the other endof the corticomotoneuronal pathway. This idea is supported by theobservation of stimulus artifact picked up by electrodes in the spinalcord. The current flowed throughout the entire pathway independent fromthe factors confounding active excitability (see introduction). Thismight cause activation of the corticomotoneuronal pathway at anypossible excitable site/s. This will ensure elicitingspike-timing-dependent plasticity that might be one of the mechanismsthat mediates the effect of the dCMS. See Dan Y, Poo M, SpikingTiming-dependent plasticity; From synapse to perception, Physiol. Rev.,86:1033-1048 (2006) for spike-timing-dependent plasticity.

In addition, the high frequency multiple spinal responses, evoked duringdCMS, can, in principle, induce long-term potentiation. Because dCMS canengage a variety of neuronal mechanisms as well as non-neuronalactivity, its effect might be a combination of many changes along thecorticomotoneuronal pathway.

The dCMS-induced enhancement of muscle force has been observed both incontrol and injured animals. The mechanisms responsible for thisamplification in these two groups of animals may overlap, but they donot have to be identical. Although, as discussed above the potentiatingeffect of dCMS could be mediated by strengthening synaptic responses,the nature and source of these changes may differ substantially in themotor pathway of control and injured animals. Axonal sprouting isprobably the primary source of synaptic connections in the damagedspinal cord. See Murray et al. supra; Bareyre et al., supra; andBrus-Ramer et al. supra. However, axonal sprouting does not grant theformation of functional connections. Therefore, one of the probablemechanisms that may mediate the potentiating effect of dCMS is therefining and strengthening of the weak synaptic connections that haveresulted from sprouting. Moreover, dormant connections that existthroughout the sensorimotor system may be activated and becomefunctional after dCMS. See Brus-Ramer M., Carmel J. B., Martin J. H.,Motor cortex bilateral motor representation depends on subcortical andinterhemispheric interactions, J. Neurosci. 29:6196-206 (2009).Potentiating the spared normal connections could also happen after dCMS.While in control animals, potentiating of normal connections andfacilitating dormant connections might be the only processes thatmediate the effect of dCMS. The results show that dCMS stimulation wasalmost twice as effective in injured animals comparing with controls.This indicates that injured spinal cord is more prone for dCMSstimulation and posses extra mechanisms mediating the dCMS effect.

In SCI animals, even before the application of dCMS, the spinalmotoneurons were responding more aggressively to cortical stimulationthan controls. Nevertheless, very weak or no muscle contraction was seen(FIG. 6). This might be due to one of two mechanisms. One would belocated in the spinal cord caudal to the lesion and/or the other being,the inexcitable peripheral nerves and/or the irresponsiveness of themuscle. Caudal to the lesion, the activity of the spinal motoneuron poolwas probably desynchronized as a result of reorganization. Supportingthis idea are the findings by Brus-Ramer and colleagues. See Brus-Rameret al. supra. Bruce-Rarner et al. reported that chronic stimulation ofcorticospinal tracts resulted in preferential axonal outgrowth towardthe ventral horn. This indicates that inter motoneuronal connections aredynamic processes, which may change by decentralization. Inexcitableperipheral axons were found in patients with SCI. See Lin C. S.,Macefield V. G., Elam M., Wallin B. G., Engel S., Kiernan M. C., Axonalchanges in spinal cord injured patients distal to the site of injury,Brain, 130:985-994 (2007). Assuming that the axons in SCI animals are insimilar conditions, they could experience an action potential failureresulting in reduced muscle contraction. Muscle atrophy is always seenin animals with SCI and humans. See, for example, Ahmed Z., WieraszkoA., Combined effects of acrobatic exercise and magnetic stimulation onthe functional recovery after spinal cord lesions, J. Neurotrauma,25:1257-1269 (2008); Liu M., Bose P., Walter C. A., Thompson F. J.,Vandenborne K., A longitudinal study of skeletal muscle following spinalcord injury and locomotor training, Spinal Cord, 46:488-93 (2008); ShahP. K., Stevens J. E., Gregory C. M., Pathare N. C., Jayaraman A., BickelS. C., Bowden M., Behrman A. L., Walter G. A., Dudley G. A., VandenborneK., Lowerextremity muscle cross-sectional area after incomplete spinalcord injury, Arch. Phys. Med. Rehabil. 87:772-778 (2006); Gordon T., MaoJ., Muscle atrophy and procedures for training after spinal cord injury,Phys. Ther. 74:50-60 (1994). This might also be one of the reasons whyspinal motoneurons responses were not translated adequately into musclecontraction.

The adequacy of motoneuronal responses was quantified by calculating thefidelity index, which is the ratio of spinal response to muscle twitchforce. The dCMS-induced changes in fidelity index were opposite incontrol and injured animals. While this index has been reduced ininjured animals, indicating improvement in the effectiveness of themotor pathway, it had increased in control animals suggesting loweringof the pathway effectiveness probably due to fatigue interference.Therefore, one can imply that injury to the spinal cord initiatesprocesses which favor regeneration of the function. The dCMS procedurelikely synchronizes and facilitates these processes, promoting recovery.

Before the dCMS application, the spontaneous activity of motoneurons inanimals with SCI was higher than that of control animals. This and theexaggerated evoked spinal responses in animals with SCI, is consistentwith the behavioral measurements that show spastic syndrome-likecharacteristics. The exaggerated spontaneous filing rate of spinalmotoneurons is also consistent with data from motor unit firing inhumans and animals after SCI and with results from intracellularrecordings from sacrocaudal motoneurons that show sustained andexaggerated firing rate in animals with SCI. See, for example, GorassiniM., Bennett D. J., Kiehn O., Eken T., Hultborn H., Activation patternsof hindlimb motor units in the awake rate and their relation tomotoneuron intrinsic properties. J. Neuro Physiol. 82:709-717 (1999);Thomas C. K., Ross B. H., Distinct patterns of motor unit behaviorduring muscle spasms in spinal cord injured subjects, J. Neuro Physiol.77:2847-2850 (1997); Harvey J. P., Gorassini M., Bennett D. J., Thespastic rat with sacral spinal cord injury in Animal model of movementdisorders, edited by Mark LeDoux, El Sevier Academic Press, 691-697(2005). Minutes after dCMS, motoneuronal spontaneous activity was stillsubstantially increased. Some of these activities were coordinated, asshown in FIG. 3B, although most of the spontaneous activity was inun-modulated pattern of firing as shown in FIG. 9A. Voltage-dependentpersistent inward currents (PICs) that strengthen synaptic inputs innormal behavior depend on descending brain-stem-released serotonin(5-HT) or noradrenaline. Here the increase in the spontaneous firingrate and the appearance of modulated activity in some animals after dCMSmay indicate better connections with brain-stem centers.

Second Experiment

The application of dCMS on humans yielded similar results. A fourteenyear old male with history of erb's palsy (right upper limb) had veryweak external rotator muscles of the shoulder. The patient had novoluntary control over these muscles and could not rotate the shoulderoutward. In addition, the shoulder external rotators were apparentlymoderately atrophied, which was determined by clinical observation. ThedCMS was applied by situating the first point negative electrode on themuscles belly (the right supraspinatus and infraspinatus muscles) andthe second point positive electrode on the contralateral muscles (samegroup). The current passed in between the two locations on oppositesides of the body, forcing the current to cross the spinal cord. It isassumed that the current follows the least resistant pathway which mostlikely involved right muscles (supraspinatus and infraspinatus muscles),the nerves innervating these muscles (the right suprascapular nerve fromC5, C6), the motor center in the spinal cord, the nerve innervatingcontralateral muscles (the left suprascapular nerve from C5, C6) andfinally the contralateral muscles (the left supraspinatus andinfraspinatus muscles), themselves. After only 15 pulses the patient wasable to rotate, with ease, the right shoulder externally, and thepatient had sensation in the arm during movement. The patient'smovement, which was gained during this treatment, persisted for at leastfour weeks.

A 14 year old female with spastic quadriplegic cerebral palsy was alsotreated with dCMS. She had significant muscle weakness throughout thefour extremities and trunk. She also had spasticity and rigidity of mostof her joints, especially the distal ones. She had extreme difficultywalking, especially climbing up and down stairs. A similar applicationof dCMS was performed on this patient. The locations of the electrodeswere varied: 1) a positive electrode was situated on the right fibularnerve and a negative electrode on the left fibular nerve. 2) a positiveelectrode on the right fibular nerve and a negative electrode on theleft median nerve. 3) a positive electrode on the left fibular nerve anda negative electrode on the right median nerve. 4) a positive electrodeon the right median nerve and a negative electrode on the left mediannerve. These configurations all allowed the current to travel across thespinal cord. After 6 sessions of the dipolar stimulation spread into twoweeks (30 minutes/session), the patient could climb 17 stepsindependently.

The above results clearly show that dCMS is an effective method thatenhances the excitability of the cortico-muscular connections in bothanimals and humans. Thus, the method of the present disclosure can beused in humans suffering after spinal cord injury, stroke, multiplesclerosis, and others. For example, the method of the present disclosurecan be employed to strengthen or awaken any weak or dormant pathway inthe nervous system as demonstrated in clinical trials.

Third Experiment

A nine-month-old child with quadriplegic paralysis due to chromosomalanomaly was treated with the same dCMS method as described in the secondexperiment. The child had been completely paralyzed without movement inthe head, the neck, the trunk, and the upper and lower extremities. Overa course of three weeks, the child was treated in four dCMS treatmentsessions that lasted 20 minutes each. After the four sessions, the childwas able to make movement in all directions in the upper extremities.She could also move her fingers in all directions and hold a toy. Shecould hold her head up and turn her head around. Further, she was ableto move her toes and lower extremities.

Fourth Experiment

Using one disc electrode situated subcutaneously over the vertebralcolumn from T10 to L1 and another at an extra-vertebral location(lateral abdominal aspect), the effects of anodal tsDC (a-tsDC) orcathodal tsDC (c-tsDC) were tested on spontaneous activity and amplitudeof cortically-elicited triceps surae (TS) muscle twitches. In adifferent set of experiments, the effects of a-tsDC or c-tsDC combinedwith rCES were tested. The data below demonstrate a unique pattern ofmodulation of corticomotoneuronal pathway activity by tsDC.

This study aimed to test whether: 1) tsDC could modulate the spontaneousactivity of spinal motoneurons in a polarity-dependent manner; 2) tsDCcould modulate corticomotoneuronal transmission; and 3) repetitivecortical stimulation (rCES) could affect spinal cord responses to tsDC.Using one disc electrode situated subcutaneously over the vertebralcolumn from T10 to L1 and another at an extra-vertebral location(lateral abdominal aspect), the effects of anodal tsDC (a-tsDC) orcathodal tsDC (c-tsDC) were tested on spontaneous activity and amplitudeof cortically-elicited triceps surae (TS) muscle twitches.

Methods

Animals

Experiments were carried out in accordance with NIH guidelines for thecare and use of laboratory animals. Protocols were approved by theCollege of Staten Island IACUC. Adult CD-1 mice (n=31) were used forthis study. Animals were housed under a 12-h light-dark cycle with freeaccess to food and water.

Surgical Procedure

Animals were anesthetized using ketamine/xylazine (90/10 mg/kg, i.p.),which has been reported to preserve corticospinal evoked potential.Anesthesia was kept at this level using supplemental dosages (˜5% of theoriginal dose) as needed, and animals were kept warm throughout theprocedure by a lamp.

The skin covering the two hindlimbs, thoracic and lumbar spines, and theskull was removed. On one side, TS muscle was carefully separated fromthe surrounding tissue, taking care to preserve the blood supply andnerves. The tendon of each of TS muscle was threaded with a hook-shaped0-3 surgical silk, which was then connected to force transducers. Tissuesurrounding the distal part of the sciatic nerve was removed. Both thesciatic nerve and TS muscle were soaked in warm mineral oil.

A craniotomy was performed to unilaterally expose the primary motorcortex (M1; usually on the right side) of the hindlimb muscles, which islocated between 0 to −1 mm from bregma and 0 to 1 mm from the midline.The dura was left intact. The exposed motor cortical area was exploredwith a stimulating electrode to locate the motor point from which thestrongest contraction of the contralateral TS muscle was obtained withthe weakest stimulus.

Electrodes

An active tsDC electrode (0.8 mm2) was situated over T10-T13; thereference electrode (Ref) was situated subcutaneously over the lateralaspect of the abdominal muscles. The surrounding tissue was removed fromthe sciatic nerve and TS muscle, and the TS muscle was connected toforce transducers. A recording microelectrode (R) was inserted into thetibial nerve. A concentric stimulating electrode (S) was placed over thecontralateral motor cortex. The spinal column and skull were rigidlysupported using a clamping system (not shown).

DC was induced through a gold surface electrode (0.8 cm2; GrassTechnologies, West Warwick, R.I., USA) situated over the vertebralcolumn from T10-L1. A similar reference electrode (0.8 cm2) was situatedover the lateral aspect of the abdominal muscles, as shown in FIG. 12. Alayer of salt-free electrode gel (Parker Laboratories, Inc., Fairfield,N.J., USA) was applied between the electrodes and the tissue. Corticalstimulation was induced by a concentric electrode (shaft diameter, 500μm; tip, 125 μm; FHC Inc., Bowdoinham, Me., USA), which was placed overthe motor cortex presentational field of the TS muscle. Extracellularrecordings were made from the TS branch of the sciatic nerve with pureiridium microelectrodes (shaft diameter, 180 μm; tip, 1-2 μm;resistance, 5.0 MΩ; WPI, Sarasota, Fla., USA). Tibial nerve potentialswere recorded from the same location (about 3 mm from the TS muscle) inall animals. The proper location was confirmed by penetration-elicitedmotor nerve spikes, which were correlated with muscle twitches.

Muscle Force Recording

The hindlimb and the proximal end of the tail were rigidly fixed to thebase of the apparatus. The knee was also fixed to the base to preventany movements from being transmitted between the stimulated muscles andthe body. The tendon of the TS muscle was attached to force displacementtransducers (FT10, Grass Technologies), and the muscle length wasadjusted to obtain the strongest twitch force (optimal length). The headwas fixed in a custom-made clamping system. Animals were kept warmduring the experiment with radiant heat.

Data Acquisition

Extracellular activity was passed through a standard head stage,amplified (Neuro Amp EX, ADInstruments, Inc., Colorado Springs, Colo.,USA), filtered (bandpass, 100 Hz to 5 KHz), digitized at 4 KHz, andstored in the computer for further processing. A power lab dataacquisition system and LabChart 7 software (ADInstruments, Inc.) wereused to acquire and analyze the data.

Polarization and Stimulation Protocols

DC was delivered by a battery-driven constant current stimulator (NorthCoast Medical, Inc., Morgan Hill, Calif., USA). A pie-test of corticalstimulation consisting of 10 pulses delivered at 1 Hz (intensity, 5.5mA; pulse duration, 1 ms) was used to elicit TS muscle twitches. Theintensity of anodal tsDC was increased in 30-s steps (0.5, 1, 1.5, 2,2.5, and 3 mA) over a total duration of 3 min. Thus, the maximal currentdensity was 3.75 A/m2 (0.003 A/0.008 m2). To avoid a stimulation breakeffect, the current intensity was ramped for 10 s. During each tsDCstep, a test (identical to the pre-test) was conducted; this test wasrepeated immediately (about 10 s) after termination of tsDC, and thenagain 5 and 20 min later. To avoid complications by excitability changesresulting from current applications, each a-tsDC and c-tsDC protocol wastested in different group of animals (n=5/group).

In addition, in two different groups of animals (n=5/group), pairedstimulation was delivered, consisting of rCES (5.5 mA, 1 ms, 1 Hz, 180pulses) combined with either a-tsDC (+2 mA) or c-tsDC (−2 mA). Apre-test and three post-tests (0, 5 and 20 mill after) of corticalstimulation (5.5 mA, 1 ms, 1 Hz, 10 pulses) were also performed.

Control Experiments

To control for possible effects of conducting the testing procedureduring tsDC, we performed experiments (n=3/group) in which only pre- andpost-tests were conducted, but no tests were performed during tsDCstimulation. The procedure was performed identically to the procedurepreviously described, in which tsDC was increased in 30-s steps. Inaddition, to control for the possible tsDC-independent effects of rCESused in a paired stimulation protocol, we also performed experiments(n=2), in which rCES (180 pulses, 1 Hz) was performed alone.

Histological Analysis

After mice were exposed to a-tsDC (n=2) or c-tsDC (n=2), segments ofspinal cord (˜1 cm) located directly below the stimulating electrodewere dissected for Hoechst stains to evaluate whether tsDC damagedspinal cord tissue. A similar spinal cord segment from an unstimulatedcontrol animal (n=1) was also analyzed. Tissues were kept overnight (4°C.) in 4% paraformaldehyde in 0.1 M PBS, then cryoprotected in 20%sucrose in PBS at 4° C. for 24 h. The spinal segments werefreeze-mounted, cut into 30 μm sections, and placed onpoly-L-lysine-coated glass slides. Sections were treated with Hoechststain (5 μg/ml; Sigma) for 30 min. then washed with PBS four times. Thesections were mounted and glass cover-slipped using mounting medium.Immunofluorescence was visualized using a Leica TCS SP2 confocalmicroscope with 405 and 488 nm lasers.

Injection of Glycine and GABA Blockers

Spinal cord segments (T13-L3) were exposed by laminectomy inanesthetized animals (n=2). The spinal column was clamped, andgastrocnemius muscles and sciatic nerves of both hindlimbs were exposed.The muscles were attached to force transducers, and recordingmicroelectrodes and stimulating electrodes were situated as shown inFIG. 12. The spinal cord was injected at the level of L3-L4 with theinhibitory neurotransmitter blockers picrotoxin and strychnine (5 μM in200 nl/2 min) using a microinjection pump (WPI, Sarasota, Fla. USA).

Calculations and Statistics

Cortically-elicited TS muscle twitches were calculated as the height ofthe twitch force relative to the baseline. The results of the pre-test,tests during tsDC, and post-tests were calculated as the average of 10responses evoked at one Hz. Spike Histogram software (ADInstrurnents,Colorado Springs, Colo., USA) was used to discriminate and analyzeextracellular spontaneous motoneuronal activity. Amplitude and frequencyof spontaneous activity were measured as the average activity during a20-s recording period before and at different points during and afterstimulation. One-way ANOVA, repeated measures ANOVA, and Kruskal-Wallisone-way ANOVA on Ranks were used to test differences between the varioustreatment conditions. Post hoc tests (Holm-Sidak method or Dunn'sMethod) were then performed to compare cortically-elicited TS twitchesat baseline or during paired stimulation with those post-stimulation. Inaddition, paired t-tests and Wilcoxon signed rank tests were used tocompare the two treatment conditions. All data are reported as groupmeans±standard error of the mean (S.E.M.). Statistical analyses wereperformed using SigmaPlot (SPSS, Chicago, Ill., USA) and LabChatsoftware (ADInstruments, Inc.) with the level of significance set atp<0.05.

Results

No morphological alterations were observed in the histochemical analysisof the spinal cold after a-tsDC or c-tsDC, as shown in FIG. 13.

1. tsDC Stimulation Modulates Spontaneous Activity of the Tibial Nerve.

To characterize the effect of tsDC on spontaneous activity of spinalneurons, firing frequency was examined before, during and after tsDC, asshown in FIGS. 14A (a-tsDC) and B (c-tsDC). As shown in FIG. 14C, a-tsDCincreased the firing frequency from a baseline of 3.3±0.3 spikes/sec to8.5±0.5, 66.5±4.9 spikes/sec, and 134.2±6.7 spikes/sec at +1, +2, and +3mA, respectively, yielding a significant effect of condition (repeatedmeasures ANOVA). Immediately following the termination of a-tsDC, thespontaneous firing frequency returned to baseline levels. As shown inFIG. 14D, c-tsDC increased the firing frequency from a baseline of2.2±0.6 spikes/sec to 6.5±3.0, 20.1±3.1 spikes/sec, and 93.1±3.8spikes/sec at −1, −2, and −3 mA, respectively, yielding a significanteffect of condition (repeated measures ANOVA). Immediately following thetermination of c-tsDC, spontaneous firing frequency returned to baselinelevels, was not statistically significantly different from baseline(p>0.05).

The a-tsDC effect on spontaneous firing frequency was significantlygreater than that of c-tsDC (Kruskal-Wallis ANOVA). Post hoc testsrevealed that all three a-tsDC intensity steps induced significantlyhigher changes in the frequency of spontaneous activity compared to thechanges induced by corresponding intensities of c-tsDC (p<0.05).

Changes in spike amplitude recorded during different intensities andpolarities of tsDC were recorded across conditions (at baseline, at eachintensity step, and after tsDC was terminated). Repeated measures ANOVAshowed a significant overall effect of condition on the amplitude ofactivity recorded during baseline (16.8±0.3 mV), which increased duringa-tsDC steps (step of +1=16.7±0.5 mV; step of +2=63.2 mV; step of+3=484.2±3.5 mV, then decreased after termination (11.9±0.7 mV), asshown in FIG. 14E. Subsequent post hoc tests showed that spike amplitudeof activity recorded during intensity steps +2 mA and +3 mA weresignificantly higher than baseline activity (p<0.05). Repeated measuresANOVA also showed a significant overall difference in the amplitude ofactivity recorded at baseline (7.0±0.3 mV), during c-tsDC (step of−1=17.3±1.5 mV; step of −2=80.4±2.2 mV; step −3=123.7±4.3 mV), and aftertermination (5.6±0.29 mV), as shown in FIG. 14F. Subsequent post hoctests showed that the amplitude of activity recorded during steps of −2mA and −3 mA was significantly higher than baseline (p<0.05).

These findings suggest that a higher intensity of tsDC can recruit morespinal neurons or potentially more classes of spinal neurons.Furthermore, the differences between amplitudes of activity recordedduring a-tsDC of +2 mA and c-tsDC of −2 mA and between a-tsDC of +3 mAand c-tsDC of −3 mA were statistically significant (t tests, p's<0.001).Overall, these findings indicate that a-tsDC and c-tsDC affect spinalneuron excitability through different mechanisms.

To further investigate the differential effects of a-tsDC and c-tsDC onspontaneous activity, we generated autocorrelograms for activity inducedby these two conditions, as well as by injection of glycine and GABAreceptor blockers. The results show tonic activity with no bursting oroscillation during a-tsDC, as shown in FIG. 15A. Conversely, c-tsDCinduced bursting, as well as oscillatory activity, as shown in FIG. 15B.Similar to c-tsDC, glycine and GABA receptor blockers induced burstingand oscillatory activity, as shown in FIG. 15C. This similarityindicates that c-tsDC and glycine and GABA receptor blockers may share amechanism of effect, which involves rhythmic-generating circuitry in thespinal cord.

2. tsDC Modulated Cortically-Elicited TS Twitches

To address whether tsDC could modulate cortically-elicited TS twitchesin an intensity- and polarity-dependent manner, TS twitches wereelicited by stimulating the motor cortex before stimulation, at fiveintensity steps during tsDC, and after stimulation (at 0, 5, and 20min). Repeated measures ANOVA, combined with post hoc tests, showed thata-tsDC affects the ability of the motor cortex to elicit TS twitches(p<0.001). Examples are shown in FIG. 16A. As shown in FIG. 16C, thebaseline average of TS twitch peak force was 0.52±0.04 g, which wasdepressed to 0.35±0.02 g, 0.32±0.01 g, 0.34±0.02 g, and 0.28±0.01 g atintensities of +1 mA, +1.5 m, +2 mA, and +2.5 mA, respectively. Incontrast, immediately after termination of a-tsDC, cortically-elicitedTS twitches were significantly improved (1.51±0.12 g), and thisimprovement persisted at 5 min (1.20±0.15 g), and at 20 min (1.9±0 38)after a-tsDC.

In the a-tsDC group, there was a main effect of group (F=19.60, p<0.001,repeated measures ANOVA), and post hocs showed that TS twitches weresignificantly weaker during intensities 1 to 2.5 mA and weresignificantly stronger at all three time points after a-tsDC, comparedto baseline. In the c-tsDC group, there was also a main effect of group(F=489.60, p<0.001, repeated measures ANOVA), and post hocs showed thatTS twitches were significantly stronger during intensities −1 to −3 mAand significantly weaker afterwards, compared to baseline. Error barsrepresent S.E.M. *p<0.05 relative to baseline.

Compared to a-tsDC, the application of c-tsDC had an opposite effect oncortically-elicited twitches. Repeated measures ANOVA, combined withpost hoc tests, showed a significant enhancement of cortically-elicitedTS twitches during c-tsDC and depression after c-tsDC. Examples areshown in FIG. 16B. As shown in FIG. 16D, the average baseline TS twitchpeak force was 0.53±0.04, which was enhanced to 1.23±0.08 g, 1.98±0.13g, 2.88±0.13 g, 4.35±0.14 g, and 5.28±0.17 g at −1 mA, −1.5 mA, −2 mA,−2.5 mA, and −3 mA, respectively. A depressive effect was seen aftertermination of c-tsDC with a peak force of 0.23±0.10 g, 0.12±0.12 g, and0.12±0.012 g at 0, 5, and 20 min, respectively. Taken together with thea-tsDC results, these data indicate that trans-spinal application ofdirect current can modulate the ability of the motor cortex to elicitactivity at the level of the lumbar spine. This modulation depends onthe polarity and intensity of the stimulation, as well as the timing oftest relative to stimulation.

3. Testing Procedure Did not Change tsDC after-Effects

To investigate a possible effect of conducting the testing procedureduring a-tsDC or c-tsDC, we repeated these experiments (n=3/group) withonly pre- and post-tests, but no tests during the tsDC stimulation. Fora-tsDC, there was no significant difference between conditions thatincluded or excluded testing during the a-tsDC stimulation (H=5.3,p=0.06, Kruskal-Wallis ANOVA). In conditions with and without testingduring stimulation, a-tsDC induced immediate improvement of TS twitches(301.14±49.33% vs. 366.9±46.9%), which persisted after 5 min(229.59±66.03% vs. 325.9±170.14%), and 20 min (387.87±117.13% vs.299.6±137.57%). Similarly, there was no effect of the testing procedureon the c-tsDC depressive after-effect (H=5.3, p>0.05, Kruskal-WallisANOVA). In conditions with and without testing during stimulation,c-tsDC depressed cortically-elicited TS twitches immediately(33.48±6.40% vs. 17.65±6.40%), after 5 mm (21.24±3.8% vs. 25.45±2.98%),and after 20 min (23.95±3.44% vs. 25.35±3.0%). These results confirmthat the testing procedure used in this study had no effect on theafter-effects induced by a-tsDC or c-tsDC.

4. Effects of a-tsDC and c-tsDC on Latency of Cortically-Elicited TibialNerve Potentials

Latency of cortically-elicited tibial nerve potentials was measuredbefore, during, and after a-tsDC and c-tsDC. Only latencies measured ata-tsDC of +2 mA and c-tsDC of −2 mA are presented because no differenceswere found between latencies at these intensities and those at otherintensities that caused significant increases in TS twitches. However,the mean latency was calculated based on measurements at all time pointsfollowing tsDC. For a-tsDC, Kruskal-Wallis ANOVA showed a significanteffect of time (baseline, during, and after stimulation), as shown inFIG. 17A. Post hoc tests revealed that the latency ofcortically-elicited tibial nerve potentials was significantly longerduring +42 mA stimulation (21.5±0.34 ms) and shorter after termination(17.92±0.21 ms) relative to baseline (19.82±0.17 ms). Similarly, forc-tsDC application, Kruskal-Wallis ANOVA showed a significant effect oftime. Post hoc tests revealed that the latency of cortically-elicitedtibial nerve potentials was significantly shorter during −2 mAstimulation (17.42±0.22 ms) and longer after termination (23.90±1.19 ms)relative to baseline (20.33±0.19 ms).

Taken together, these data indicate that tsDC affects the excitabilityof spinal neurons in a way that changes their ability to respond to themotor cortex. Thus, changes in latency may be due to redirection of theintra-spinal pathway to a faster or slower route depending on the numberof synapses or simply due to changes in the recruitment pattern ofspinal neurons.

5. Paired rCES and tsDC Stimulation

The motor cortex was stimulated for 3 min (180 pulses, 1 Hz, maximalintensity ˜5.5 mA) during either a-tsDC (+2 mA) or c-tsDC (−2 mA), asshown in FIGS. 18A and 18B. Paired rCES and a-tsDC was associated with asignificant improvement in cortically-elicited TS twitches aftertermination of stimulation (0.80±0.10 g) compared to baseline (0.39±0.05g) (p<0.001), as shown in FIG. 18C. Notably, paired rCES and c-tsDCshowed a similar improvement after termination (3.67±0.51 g) compared tobaseline (0.21±0.51 g) (p<0.001), as shown in FIG. 18D. Improvementfollowing those two different stimulation paradigms persisted with nonotable change immediately, at 5 min and at 20 min after termination.Thus, results presented after termination represent the average of thesethree time points. The effect of rCES alone was tested in separate groupof animals (n=2), and no change was found after termination compared tobaseline (t test, p>0.05) (data not shown).

A total of four stimulation paradigms used in the current experimentaffected cortically-elicited TS contraction: a-tsDC, c-tsDC, a-tsDC withrCES, and c-tsDC with rCES. Kruskal-Wallis ANOVA showed a significanteffect of condition (H=66.97, p<0.001). Multiple comparisons showed thatpaired c-tsDC and rCES was more effective than all other paradigms(2287.07±342.49%) (p<0.05), especially for reversing the depressiveeffect seen after c-tsDC (33.66±9.82%). Paired a-tsDC and rCES showed nosignificant difference (252.88±30.79%) compared to a-tsDC alone(329.18±38.79%) (p>0.05). These findings indicate that cortical activityhad a strong influence on c-tsDC after-effects, however, it had noinfluence on a-tsDC after-effects.

Discussion

Histological analysis demonstrated no harmful morphological effects ofthe tsDC parameters used in the present study. The maximal currentdensity used was 3.75 A/m² for a duration of 3 min, which is much lowerthan the range typically used in tats and mice as known in the art. Inthis study, spinal cord stimulation differed from cranial stimulation inthree respects: (1) the distance from the electrode surface to theventral aspect of the spinal cord was ˜7 mm, as opposed to the distanceto the cranium of ˜0.3 mm; (2) bone, muscle and fat tissue was presentbetween the electrode and spinal cord, while only bone was present atthe cranium; and (3) the volume of the conductor surrounding the targettissue was much larger in the spinal cord than in the brain, potentiallydeforming the current and reducing its density.

Both a- and c-tsDC markedly increased the frequency and amplitude ofspontaneous tibial nerve activity in an intensity-dependent fashion.Interestingly, a-tsDC was more effective than c-tsDC in increasingfiring frequency and recruiting units with larger amplitude. Theseresults are in agreement with data from a-tsDC stimulation of thecerebral cortex, hippocampal slices, and cerebellum. The effects ofc-tsDC on neuronal discharges were more complex in three respects.First, c-tsDC only caused significant changes at higher intensities (−2and −3 mA). Second, c-tsDC did not cause firing of neurons with largespikes, but was observed in some experiments to inhibit firing of largespikes (1 mV), while increasing firing of smaller spikes. Third, as seenin FIG. 14B, c-tsDC evoked rhythmic firing. The c-tsDC-induced increasein firing rate supports previous observations in which negative currentsoccasionally increased firing rate. See Bindman L. J., Lippold O. C.,and Redfearn J. W., The action of brief polarizing currents on thecerebral cortex of the rat (1) during current flow and (2) in theproduction of long-lasting after-effects, J. Physiol. 172: 369-382(1964).

During stimulation, a-tsDC depressed cortically-elicited TS twitches,while c-tsDC markedly potentiated twitches. From immediately aftertermination of tsDC until at least 20 min later, cortically-elicited TStwitches were markedly potentiated after a-tsDC and depressed afterc-tsDC. Moreover, while a-tsDC increased the latency ofcortically-elicited tibial nerve potentials, c-tsDC decreased thislatency. After a-tsDC or c-tsDC stimulation was terminated, the effecton latency was reversed.

Changes in latency were observed despite a steady intensity of corticalstimulation, suggesting that factors underlying these changes are notlikely to include the switch from a cortical site of activation to adeeper location (Rothwell et al. 1994). Instead, these factors mayinclude: (1) axonal hyperpolarization (Moore and Westerfield 1983) byc-tsDC or (2) activating preferential spinal circuits that mediatecorticomotoneuronal transmission. In rodents, the corticomotoneuronalpathway has two indirect routes, a faster route mediated viareticulospinal neurons and a slower route mediated via segmentalinterneurons. The present findings suggest that c-tsDC may shift thepattern of excitability at the spinal cord toward the fasterreticulospinal route. Interestingly, pairing a-tsDC with rCES (1 Hz)potentiated cortically-elicited TS twitches, but was not different froma-tsDC alone. Conversely, pairing c-tsDC with rCES potentiatedcortically-elicited TS twitches and had the greatest effects of anystimulation condition.

The differences in the effects of a-tsDC and c-tsDC on neuronal activitysuggest that the two conditions affect distinctive neuronal typesthrough different mechanisms. The topography of spinal neurons relativeto the direction of current determines the current locus and type ofeffect (i.e., increase or decrease in excitability). As illustrated inFIG. 19, a dorsal cathodal current should depolarize neuronalcompartments closer to the electrode and hyperpolarize compartmentsfarther from the electrode. Thus, an interneuron with its dendrites andsoma at the ventral aspect of the spinal cord and its axon at the dorsalaspect would have a hyperpolarized dendritic tree and soma and adepolarized axon and nerve terminal. Such a neuron would be lessresponsive to synaptic activation, but would have a lower threshold tospontaneously fire an axonally-generated action potential. A spinalneuron oriented in the opposite direction would show an oppositeresponse to cathodal stimulation. This argument is supported by thefinding that motoneuron responses to dorsolateral and medial funiculusstimulation were facilitated by depolarizing currents in the dendritesand soma, but were not affected by hyperpolarizing currents, which havealso been shown to occur in the hippocampus (Bikson 2004). SeeDelgado-Lezama R., Perrier J. F., and Hounsgaard J., Local facilitationof plateau potentials in dendrites of turtle motoneurones by synapticactivation of metabotropic receptors, J. Physiol. 515 (Pt 1): 203-207(1999) and Bikson M., Effects of uniform extracellular DC electricfields on excitability in rat hippocampal slices in vitro, J. Physiol.557: 175-190 (2004).

Presynaptic depolarization has been shown to decrease presynaptic nerveaction potentials and EPSPs. See Hubbard J. I. and Willis W. D., Theeffects of depolarization of motor nerve terminals upon the release oftransmitter by nerve impulses, J. Physiol. 194: 381-405 (1968); HubbardJ. I. and Willis W. D., Reduction of transmitter output bydepolarization, Nature 193: 1294-1295 (1962). The decrease in thepresynaptic nerve action potentials and EPSPs may play a role indepressing cortically-elicited TS twitches during a-tsDC. In addition,hyperpolarization of the soma and dendrites could depress motoneuronresponses to cortical stimulation during a-tsDC. Alternativeexplanations could include: (1) increased numbers of refractory motorneurons due to increased spontaneous firing, or (2) preferentialactivation of the spinal or supraspinal inhibitory pathway.

Rhythmic activity was observed during c-tsDC but not a-tsDC, indicatingthat c-tsDC may have a depressive effect on spinal inhibitoryinterneurons. Such interneurons might be inhibited because of theirtopography relative to the applied electrical field. C-tsDC mighthyperpolarize both excitatory and inhibitory spinal interneurons. If itis assumed that inhibitory and excitatory spinal interneurons containdifferent membrane channels (e.g., fewer low-voltage-activated T-typecalcium channels and hyperpolarization-activated cation channels ininhibitory interneurons), then hyperpolarization would silenceinhibitory interneurons, hence disinhibiting the excitatoryinterneurons. In contrast, in spinal rhythmogenic neurons,hyperpolarizing tsDC might activate the hyperpolarization-activated,nonselective cation current (Ih). In combination with T-type Cachannels. Ih should gradually depolarize the cell membrane to reach thethreshold for an action potential, which could be another mechanismmediating c-tsDC-induced potentiation of cortically-elicited TStwitches.

Moreover, cathodal stimulation has been shown to increase theexcitability of axons aligned perpendicular to the direction of current.See Ardolino G., Bossi B., Barbieri S., and Priori A., Non-synapticmechanisms underlie the after-effects of cathodal transcutaneous directcurrent stimulation of the human brain, J. Physiol. 568: 653-663 (2005).Therefore, in the present study, the corticospinal tract, which passesbelow the cathodal electrode, would be expected to increase axonalexcitability and hence spinal output. Conversely, the dendrites and somaof motoneurons would be hyperpolarized and axons would be depolarized inresponse to a-tsDC stimulation. Axonal depolarization at locations thataffect voltage-sensitive membrane conductances could increase the firingrate and amplitude of spontaneous activity during a-tsDC.

In the spinal cord, L-type Ca+2 channels present in motoneuron dendritesmediate the facilitatory action of depolarizing currents. However, theexact cellular mechanisms mediating DC stimulation after-effects are notclear. Notably, mechanisms mediating the depressive after-effects ofcathodal DC stimulation are completely unknown. We suggest that thepattern of c-tsDC-induced polarization (e.g., pre-synaptichyperpolarization and post-synaptic depolarization) might activatedepression-mediating mechanisms, such as retrograde signaling byendocannabinoids that selectively depresses inhibitory pre-synapticterminals.

While the invention has been described in terms of specific embodiments,it is evident in view of the foregoing description that numerousalternatives, modifications and variations will be apparent to thoseskilled in the art. Accordingly, the invention is intended to encompassall such alternatives, modifications and variations which fall withinthe scope and spirit of the invention and the following claims.

What is claimed is:
 1. A method of improving a neuromuscular condition of a vertebrate being, said method comprising: placing at least one active electrode at, or in proximity to, a first point and at least one reference electrode at, or in proximity to a second point, wherein said first point is located at the central nerve system of a vertebrate being, and said second point is located outside the central nervous system of said vertebrate being; and enhancing a neural connection between said first point and said second point by passing electrical current between said at least one active electrode and said at least one reference electrode.
 2. The method of claim 1, wherein said first point is located at a spinal column of said vertebrate being.
 3. The method of claim 2, wherein said electrical current is provided by a stimulator including at least one stimulator unit that applies electrical pulses across said at least one active electrode and said at least one reference electrode.
 4. The method of claim 2, further comprising placing at least another reference electrode at, or in proximity to, an additional point located outside the central nervous system of said vertebrate being.
 5. The method of claim 4, wherein said vertebrate being is a human, and said second point and said additional point are located at the pelvis of said human.
 6. The method of claim 2, further comprising applying an additional electrical stimulus to the motor cortex of said vertebrate being during passing of said electrical current.
 7. The method of claim 6, wherein said additional electrical stimulus is a local stimulus in the form of at least one electrical pulse.
 8. The method of claim 7, wherein said at least one electrical pulse is applied synchronously with said passing of said electrical current.
 9. The method of claim 2, further comprising applying an additional electrical stimulus to at least one muscle of said vertebrate being during passing of said electrical current.
 10. The method of claim 9, wherein said additional electrical stimulus is a stimulus in the form of at least one electrical pulse.
 11. The method of claim 10, wherein said at least one electrical pulse is applied synchronously with said passing of said electrical current.
 12. The method of claim 2, further comprising: applying a first additional electrical stimulus to the motor cortex of said vertebrate being during passing of said electrical current; and applying a second additional electrical stimulus to at least one muscle of said vertebrate being during passing of said electrical current.
 13. The method of claim 12, wherein said first additional electrical stimulus is a local stimulus in the form of at least one first electrical pulse, and said second additional electrical stimulus is a stimulus in the form of at least one second electrical pulse.
 14. The method of claim 13, wherein said at least one first electrical pulse and said at least one second electrical pulse are applied synchronously with said passing of said electrical current.
 15. The method of claim 13, wherein said at least one first electrical pulse and said at least one second electrical pulse are applied asynchronously from said passing of said electrical current.
 16. The method of claim 1, wherein said electrical current is passed as a plurality of pulses, wherein each of said plurality of pulses has a duration from 0.5 ms to 5 ms.
 17. The method of claim 1, wherein said electrical current is passed a plurality of pulses having a frequency from 0.5 Hz to 5 Hz.
 18. The method of claim 1, further comprising providing a prompt to move a limb to said vertebrate being during, or immediately before, said passing of said electrical current.
 19. The method of claim 18, wherein said prompt is an aural prompt, a visual prompt, or a tactile prompt.
 20. The method of claim 18, wherein said vertebrate being is a human, and said prompt is provided by another human to said human.
 21. The method of claim 18, wherein said prompt is provided by an automated control unit configured to generate said prompt in synchronization with said passing of said electrical current.
 22. The method of claim 1, wherein said vertebrate being is a mammal, and second points is located at a muscle in a limb of said mammal.
 23. The method of claim 1, wherein said vertebrate being is a human, and second points is located at a muscle in a human limb.
 24. The method of claim 1, wherein said vertebrate being is a human with at least one condition selected from an injury suffered at a location in the spinal column, cerebral palsy, amyotrophic lateral sclerosis, traumatic brain injury, stroke, peripheral palsy, Erb's palsy, sciatica, and other peripheral nerve injuries due to nerve compression, tension, or torsion, and wherein said enhancing of said neural connection alleviates or reduces said at least one condition.
 25. A method of improving a neuromuscular condition of a vertebrate being, said method comprising: placing at least one active electrode at, or in proximity to, a first point located on one side of a spinal column of a vertebrate being and at least one reference electrode at, or in proximity to, a second point, wherein said first point is located on the opposite side of said spinal column, wherein locations of said first point and said second point are independently selected from the motor cortex and a muscle of said vertebrate being; and enhancing a neural connection between said first point and said second point by passing electrical current between said at least one active electrode and said at least one reference electrode.
 26. The method of claim 25, wherein at least one path of said electrical current runs across said spinal column and between said first point and said second point.
 27. The method of claim 26, wherein said at least one path of said electrical current includes a motor pathway between said motor cortex and a muscle.
 28. The method of claim 27, wherein said first point is a point at the motor cortex and said second point is a point at a muscle.
 29. The method of claim 27, wherein said second point is a point at the motor cortex and said first point is a point at a muscle.
 30. The method of claim 25, wherein said first point is a point at a first muscle, and said second point is a point at a second muscle that is different from said first muscle and located on the opposite side of said spinal column from said first muscle.
 31. The method of claim 30, wherein said at least one path of said electrical current includes at least one first lower motoneuron connected to said first point and at least one second lower motoneuron connected to said second point.
 32. The method of claim 25, wherein said at least one active electrode is a single active electrode, and said at least one reference electrode is a single reference electrode.
 33. The method of claim 25, wherein said at least one active electrode is a plurality of active electrodes or said at least one reference electrode is a plurality of reference electrodes.
 34. The method of claim 25, wherein each of said at least one active electrode and said at least one reference electrode is attached to said motor cortex or a muscle topically, underneath a skin, or by surgical implantation.
 35. The method of claim 25, further comprising identifying a motoneuron that affects movement of a muscle of said vertebrate being in said spinal column, wherein said muscle is subsequently attached to one of said at least one active electrode or said at least one reference electrode.
 36. The method of claim 34, further comprising: determining a maximal stimulus strength for said motoneuron at which no further increase in muscle contraction of said muscle is observed with an increase in strength of electrical stimulation to said motoneuron; and setting a voltage differential between at least one active electrode and said at least one electrode during said passing of said current in proportion to said determined maximal stimulus strength.
 37. The method of claim 36, wherein said voltage differential is set at a same voltage as said maximal stimulus strength.
 38. The method of claim 25, wherein said electrical current is passed as a plurality of pulses, wherein each of said plurality of pulses has a duration from 0.5 ms to 5 ms, and said electrical current is passed a plurality of pulses having a frequency from 0.5 Hz to 5 Hz.
 39. The method of claim 25, further comprising providing a prompt to move a limb to said vertebrate being during, or immediately before, said passing of said electrical current.
 40. The method of claim 39, wherein said prompt is an aural prompt, a visual prompt, or a tactile prompt.
 41. The method of claim 39, wherein said prompt is provided by an automated control unit configured to generate said prompt in synchronization with said passing of said electrical current.
 42. The method of claim 25, wherein said vertebrate being is a human with at least one condition selected from an injury suffered at a location in the spinal column, cerebral palsy, amyotrophic lateral sclerosis, traumatic brain injury, stroke, peripheral palsy, Erb's palsy, sciatica, and other peripheral nerve injuries due to nerve compression, tension, or torsion, and wherein said enhancing of said neural connection alleviates or reduces said at least one condition.
 43. A system for improving a neuromuscular condition of a vertebrate being, said system comprising: at least one active electrode, each sized and configured to be placed at, or in proximity to, a first point located at the central nerve system of a vertebrate being; at least one reference electrode, each sized and configured to be placed at, or in proximity to, a second point that is located outside the central nervous system of said vertebrate being; a stimulator unit configured to generate electrical stimulation waveforms; and at least one first lead wire that couples said stimulator unit to said at least one active electrode and at least one second lead wire that couples said stimulator unit to said at least one reference electrode, wherein said system is configured to form a current path through a motor pathway across said spinal column between said first point and said second point.
 44. The system of claim 43, wherein each of said at least one active electrode is sized and configured to be placed at, or in proximity to, a spinal column of said vertebrate being.
 45. The system of claim 44, wherein said at least one active electrode and said at least one reference electrode are sized and configured to be placed at, or in proximity to, the spinal column of a human.
 46. The system of claim 43, further comprising at least another active electrode and at least another reference electrode, each sized and configured to be placed at, or in proximity to, the motor cortex of said vertebrate being.
 47. The system of claim 46, wherein said at least another active electrode and said at least another reference electrode are sized and configured to be placed at, or in proximity to, the motor cortex of a human.
 48. The system of claim 46, further comprising another stimulator unit configured to generate additional electrical stimulation waveforms that are applied across said at least another active electrode and said at least another reference electrode.
 49. The system of claim 48, wherein said stimulator unit and said other stimulator unit are synchronized to provide said electrical stimulation and said additional electrical stimulation simultaneously.
 50. The system of claim 43, further comprising at least another active electrode and at least another reference electrode, each sized and configured to be placed at, or in proximity to, a muscle of said vertebrate being.
 51. The system of claim 50, wherein said at least another active electrode and said at least another reference electrode are sized and configured to be placed at, or in proximity to, a muscle of a human.
 52. The system of claim 50, wherein said stimulator unit and said other stimulator unit are synchronized to provide said electrical stimulation and said additional electrical stimulation simultaneously.
 53. The system of claim 43, further comprising prompt means for providing a prompt to move a limb to said vertebrate being during, or immediately before, said passing of said electrical current.
 54. A system for improving a neuromuscular condition of a vertebrate being, said system comprising: at least one active electrode, each sized and configured to be placed on, or in proximity to, a first point that is selected from the motor cortex and a muscle and is located on one side of a spinal column of a vertebrate being; at least one reference electrode, each sized and configured to be placed on, or in proximity to, a second point that is selected from said motor cortex and a muscle and is located on the opposite side of said spinal column; a stimulator unit configured to generate electrical stimulation waveforms; and at least one first lead wire that couples said stimulator unit to said at least one active electrode and at least one second lead wire that couples said stimulator unit to said at least one reference electrode, wherein said system is configured to form a current path through a motor pathway across said spinal column between said first point and said second point.
 55. The system of claim 54, wherein one of said at least one active electrode and said at least one reference electrode is sized and configured to be placed on, or in proximity to, said motor cortex.
 56. The system of claim 55, wherein said one of said at least one active electrode and said at least one reference electrode is sized and configured to be placed on, or in proximity to, the motor cortex of a mammal having limbs.
 57. The system of claim 55, wherein said one of said at least one active electrode and said at least one reference electrode is sized and configured to be placed on, or in proximity to, the motor cortex of a human.
 58. The system of claim 54, wherein all of said at least one active electrode and said at least one reference electrode are sized and configured to be placed on, or in proximity to, a muscle of said vertebrate being.
 59. The system of claim 58, wherein all of said at least one active electrode and said at least one reference electrode are sized and configured to be placed on, or in proximity to, a muscle in a limb of a mammal having limbs.
 60. The system of claim 58, wherein said one of said at least one active electrode and said at least one reference electrode is sized and configured to be placed on, or in proximity to, a human limb.
 61. The system of claim 54, wherein said at least one active electrode is a single active electrode, and said at least one reference electrode is a single reference electrode.
 62. The system of claim 54, wherein said at least one active electrode is a plurality of active electrodes or said at least one reference electrode is a plurality of reference electrodes.
 63. The system of claim 54, wherein each of said at least one active electrode and said at least one reference electrode is configured for attachment to the motor cortex or a muscle of said vertebrate being topically, underneath a skin, or by surgical implantation.
 64. The system of claim 54, further comprising at least one probe for identifying a motoneuron that affects movement of a muscle of said vertebrate being and located in said spinal column by applying electrical voltage thereto.
 65. The system of claim 54, further comprising prompt means for providing a prompt to move a limb to said vertebrate being during, or immediately before, said passing of said electrical current. 